Compositions and methods for capture and elution of biological materials via particulates

ABSTRACT

Lysing may include agitating a specimen in a chamber along with a medium that includes a particulate lysing material that has an affinity for a biological material. Lysing material may include beads or other material which may be coated that facilitates binding. The medium may include a fluid with a high salt or low pH level. Binding of biological materials to solid surfaces may be induced by particular media. The biological material may be eluted by lowering a concentration of salt or increasing a pH level. Lysing materials with two or more different affinities may be employed. Lysing materials may include particles of different sizes. Heating may be used. Lysing may be performed in a flow through apparatus. Surfaces of solid materials may be modified to capture bacteria with high cell wall lipid content.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. 119(e) to U.S.provisional patent application Ser. No. 61/427,045 filed Dec. 23, 2010;and U.S. provisional patent application Ser. No. 61/444,607 filed Feb.18, 2011, each of which are incorporated herein by reference, in theirentirety.

TECHNICAL FIELD

The present disclosure relates to extraction, capture and elution ofbiological material, for example nucleic acids such as DNA, frombiological specimens using particulate materials. The present disclosurealso relates to lysing and in particular to systems, apparatus andmethods to perform lysing of a biological material to be lysed using alysing particulate material.

BACKGROUND

Lysis of biological specimens, for example cell lysis, is used toprovide biological materials for compositional analysis. Specificbiological materials may include proteins, lipids, and nucleic acidseither individually or as complexes. When a cell membrane is lysed,certain organelles—nuclei, mitochondria, lysosomes, chloroplasts, and/orendoplasmic reticulum—may be isolated. Such may be analyzed usingmethods such as polymerase chain reaction (PCR), electron microscopy,Western blotting or other analysis techniques.

There are numerous approaches to performing lysis. For example,enzymatic approaches may be employed to remove cell walls usingappropriate enzymes in preparation for cell disruption or to prepareprotoplasts. Another approach employs detergents to chemically disruptcell membranes. These chemical approaches may adversely affect theresulting product, for example degrading the bio-products beingreleased. Consequently, chemical approaches may, in some instances, notbe practical.

Yet another approach employs ultrasound to produce cavitation andimpaction for disrupting the cells. Such an approach may not achieve ashigh a lysis efficiency as may be required or desired for manyapplications.

Yet still another approach employs beads (e.g., glass or ceramic) whichare agitated, for example, via a vortex mixer. Such an approachsuccessfully addresses the issues raised by chemical lysis approaches,yet improvements in such an approach are desirable.

Particular biological materials that are isolated from the interior ofcells or viruses for use in a variety of analysis or testing proceduresinclude nucleic acids. These may be isolated and used, for example, intesting for bacterial or viral infections. Nucleic acid analysis ortesting for such purposes may provide improved sensitivity or mayshorten the time between incidence of an infection and appearance of apositive test, compared to results obtained from more traditionalantibody testing. Nucleic acid analysis or testing typically involvesextraction and isolation of a nucleic acid of interest, e.g.,deoxyribonucleic acid (DNA), from the biological specimens, followed byamplification reactions, such as PCR. Amplification of the isolatednucleic acid increases the sensitivity of detection and identificationof the resulting nucleic acid.

Commonly used techniques for rapid extraction and isolation of nucleicacids, in particular DNA, from cells utilize membranes or beads,including magnetic beads, that are made from silica or from othermaterials that capture DNA nonspecifically on the basis of thepolyanionic chemistry of DNA. Most such techniques rely on the use ofharsh reagents, such as chaotropic salts, e.g., guanidiniumhydrochloride, guanidinium thiocyanate, or proteases, to lyse cells tofree the DNA. The harsh reagents used in such methods of DNA isolationare not compatible with subsequent amplification reactions. The reagentsmust thus be thoroughly removed, often by numerous wash steps, prior toelution and subsequent use or analysis of the isolated DNA. Even thewash steps may include reagents that are incompatible with subsequentreactions and must thus be removed. For example, washes may includealcohol which must then be removed by evaporation. Additionally, methodsfor isolating biological materials of interest, such as DNA, mayco-isolate other biological materials that may interfere with subsequentanalyses. Approaches that eliminate such manipulations, particularly theuse and removal of harsh reagents or co-isolation of undesirablebiological materials as contaminants in the products, couldadvantageously improve the efficiency of the process and the utility ofthe isolated product, and could thus simplify and optimize processing ofcell-contained substances, in particular DNA, for such purposes.

BRIEF SUMMARY

There is a need for particle-based or other solid phase systems andmethods that efficiently obtain biological material. Such improvedsystems and methods may reduce the amount of time required to process asample (i.e., a sample from which to obtain the biological material)and/or to increase throughput. Such may also increase the degree ofthoroughness of obtaining the material, yielding greater amounts ofmaterial from a given sample size. There is also a need for systems andmethods for lysis, capture and elution of biological material withoutseparate processing of particles on which the biological material iscaptured. In particular, there is a need for such systems and methodsthat allow lysis, capture and elution of biological materials within thesame system by simply controlling chemical composition and flow ofreagents within the system. There is also a need for systems and methodsto lyse cells without use of harsh reagents. Such may avoid wash stepsduring processing of biological material captured by particle-basedsystems. There is also a need for methods and formulations for moreefficiently processing particle-bound or other solid phase-boundbiological materials produced by standard methods known in the art.There is also a need for solid phase materials and methods for specificcapture of cells or cell components. For example, there is a need formaterials and methods for efficiently isolating and processingmicroorganisms having cell walls with high lipid content, such asmycobacteria. There is also a need for materials and methods for moreefficient and effective removal of contaminating substances frombiological materials isolated for further analysis. For example, thereis a need for materials and methods to remove biological contaminantsthat may interfere with subsequent analysis or may otherwise limit thesensitivity of analysis of biological materials of interest, such asDNA. There is also a need for systems and methods that may allow sampleheating integrated within a system. There is also a need for systems andmethods for specific capture of cell components. There is also a needfor lysing equipment that is small and hence portable, and that isrelatively inexpensive yet sufficiently robust to withstand travel orharsh operating environments.

A method of isolating nucleic acid may be summarized as includingcontacting a specimen containing a nucleic acid with a particulatematerial having an affinity for the nucleic acid to allow at least aportion of the nucleic acid to bind to the particulate material; andwashing the particulate material having the bound nucleic acid with alow ionic strength zwitterion-containing buffer to yield a washedparticulate material having the nucleic acid bound thereto. Contacting aspecimen containing a nucleic acid with a particulate material mayinclude contacting the specimen with a plurality of particles comprisingat least one of a ceramic, a glass, a zirconia, a silica, a sand, or ametal core coated by a material that facilitates binding of the nucleicacid. The specimen may include a binding medium and contacting aspecimen containing a nucleic acid with a particulate material mayinclude contacting the specimen including the binding medium with theparticulate material. The binding medium may include a composition thatinduces binding of the nucleic acid to the particulate material. Thecomposition that may induce binding of the nucleic acid to theparticulate material may include one or more of a salt concentrationgreater than or equal to 2 molar, a chaotropic substance, and analcohol. Washing the particulate material with a low ionic strengthzwitterion-containing buffer may include washing the particulatematerial with a low ionic strength zwitterion-containing buffer having apH between about pH 3 and about pH 6. Washing the particulate materialwith a low ionic strength zwitterion-containing buffer may includewashing the particulate material with a buffer comprising one or more ofan amino acid, an aminosulfonic acid, or an aminocarboxylic acid.Washing the particulate material with a low ionic strengthzwitterion-containing buffer may include washing the particulatematerial with a buffer comprising at least one zwitterionic substancehaving a pKa within a range between about 2 and about 4. The low ionicstrength zwitterion-containing buffer may be a glycine buffer at aboutpH 4.

The method may further include applying to the washed particulatematerial a low ionic strength buffer having a pH that will adjust the pHat the surface of the particulate material above at least about 6 toelute the nucleic acid from the particulate material.

A formulation for use in isolating nucleic acid by a particulatematerial may be summarized as including a low ionic strengthzwitterion-containing buffer having a pH less than about 6 to inducebinding of nucleic acid to or to prevent release of bound nucleic acidfrom the particulate material. The low ionic strengthzwitterion-containing buffer may have a pH between about 3 and about 6.The low ionic strength zwitterion-containing buffer may include azwitterionic substance having a pKa between about 2 and about 4. The lowionic strength zwitterion-containing buffer may include a zwitterionicsubstance having a pKb between about 9 and about 11. The low ionicstrength zwitterion-containing buffer may include a zwitterionicsubstance that is an amino acid, an aminosulfonic acid, or anaminocarboxylic acid. Low ionic strength zwitterion-containing buffermay be a glycine buffer having a pH about 4.

A kit for use in isolating nucleic acid may be summarized as including aparticulate material that has an affinity for nucleic acid; a low ionicstrength zwitterion-containing buffer having a pH less than about 6; andinstructions for use of the kit to isolate nucleic acid. The low ionicstrength zwitterion-containing buffer may have a pH between about 3 andabout 6. The low ionic strength zwitterion-containing buffer may includea zwitterionic substance that is an amino acid, an aminosulfonic acid,or an aminocarboxylic acid.

A method of isolating phosphate-containing polyanions may be summarizedas including contacting a specimen having a phosphate-containingpolyanion with an amine-containing solid phase material having anaffinity for the phosphate-containing polyanion to allow at least aportion of the phosphate-containing polyanion to bind to the solid phasematerial; and applying to the amine-containing solid phase material aformulation comprising phosphate-containing anions to elute thephosphate-containing polyanion from the amine-containing solid phasematerial. The phosphate-containing polyanion may be a nucleic acid andcontacting a specimen having a phosphate-containing polyanion includescontacting the specimen having the nucleic acid present therein. Theformulation including phosphate-containing anions may include one ormore types of nucleoside phosphate. The formulation includingphosphate-containing anions may include one or more types of nucleosidetriphosphate. The one or more types of nucleoside triphosphate mayinclude at least one of adenosine triphosphate, guanosine triphosphate,cytidine triphosphate, uridine triphosphate, deoxyadenosinetriphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate,or deoxythymidine triphosphate. The solid phase material may include atleast one of a particulate, a bead, a membrane, a flow channel, or atube.

A kit for use in isolating phosphate-containing polyanions from aspecimen may be summarized as including an amine-containing solid phasematerial having an affinity for phosphate-containing polyanions; aformulation having phosphate-containing anions; and instructions for useof the kit to isolate phosphate-containing polyanions from a specimen.The formulation may include one or more nucleoside phosphates. Theformulation may include one of more nucleoside triphosphates. Theformulation may include at least one of adenosine triphosphate,guanosine triphosphate, cytidine triphosphate, uridine triphosphate,deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidinetriphosphate, or deoxythymidine triphosphate. The amine-containing solidphase material may include at least one of a particulate, a bead, amembrane, a flow channel, or a tube.

A method of isolating a sulfonated polyanions may be summarized asincluding contacting a specimen having the sulfonated polyanion with anamine-containing solid phase material having an affinity for thesulfonated polyanion to allow at least a portion of the sulfonatedpolyanion to bind to the solid phase material; and applying to theamine-containing solid phase material a formulation comprisingsulfate-containing anions to elute the sulfonated polyanion from theamine-containing solid phase material.

A kit for use in isolating sulfonated polyanions from a specimen may besummarized as including an amine-containing solid phase material havingan affinity for sulfonated polyanions; a formulation havingsulfate-containing anions; and instructions for use of the kit toisolate sulfonated polyanions from a specimen.

A method of capturing microorganisms having high cell wall lipid contentmay be summarized as including modifying a surface of a material toinduce binding of microorganisms having a high cell wall lipid content;and contacting a specimen that contains microorganisms having the highcell wall lipid content with the material having the modified surface tocapture the microorganisms having high cell wall lipid content on themodified surface of the material. Modifying a surface of a material toinduce binding of microorganisms having a high cell wall lipid contentmay include reacting the surface of the material with one or morereagents to yield a hydrophobic surface.

The method may further include lysing the captured microorganisms havinghigh cell wall lipid content to release nucleic acids from the capturedmicroorganisms; and recovering the nucleic acids released from thecaptured microorganisms. Lysing the captured microorganisms may includelysing the microorganisms in a formulation having low to moderate ionicstrength. Recovering the nucleic acids released from the capturedmicroorganisms may include binding the nucleic acids to the materialmodified to induce binding of microorganisms and eluting the nucleicacids from the material. Modifying the surface of the material to inducebinding of microorganisms having a high cell wall lipid content mayinclude reacting the surface of the material with one or more reagentsto yield a hydrophobic and positively charged surface. Reacting thesurface of the material with one or more reagents to yield a hydrophobicand positively charged surface may include reacting the surface of thematerial with polydiallyldimethylammonium chloride.

The method may further include lysing the captured microorganisms havinghigh cell wall lipid content to release nucleic acids from the capturedmicroorganisms; and recovering the nucleic acids released from thecaptured microorganisms. Lysing the captured microorganisms may includelysing the microorganisms in a formulation containing one or morenucleoside triphosphates to limit binding of nucleic acids to thehydrophobic and positively charged surface of the material. Modifyingthe surface of the material to induce binding of microorganisms having ahigh cell wall lipid content may include reacting the surface of thematerial with one or more reagents to yield a hydrophobic and negativelycharged surface. Contacting a specimen that contains microorganismshaving a high cell wall lipid content with the surface of the modifiedmaterial may include contacting the specimen with the surface in aformulation containing one or more of a high salt concentration, a lowpH, and divalent cations.

The method may further include lysing the captured microorganisms havinghigh cell wall lipid content to release nucleic acids from the capturedmicroorganisms; and recovering the nucleic acids released from thecaptured microorganisms.

The method may further include washing the captured microorganisms in aformulation having low ionic strength to limit binding of nucleic acidsto the hydrophobic and negatively charged surface of the material beforethe lysing. Modifying the surface of the material to induce binding ofmicroorganisms having a high cell wall lipid content may includereacting the surface of the material with one or more silane reagents.Reacting the surface of the material with one or more silane reagentsmay include reacting the surface with at least one silane reagentselected from alkyl silanes, vinyl silanes, or amino silanes. Thematerial having a surface modified to induce binding of microorganismshaving high cell wall lipid content may include at least one of aparticulate, a bead, a membrane, a flow channel, or a tube. The materialhaving a surface modified to induce binding of microorganisms havinghigh cell wall lipid content may include at least one of a ceramic, aglass, a silica, or a sand. The material having a surface modified toinduce binding of microorganisms having high cell wall lipid content mayinclude at least one of a gold particulate, a gold bead, a gold-coatedparticulate, or a gold-coated bead.

The method of may further include amplifying the recovered nucleic acidsby an amplification reaction; and detecting the nucleic acids amplifiedby the amplification reaction. The microorganisms having high cell walllipid content may include mycobacteria.

A solid phase material for binding microorganisms having high cell walllipid content may be summarized as including a hydrophobic surface toinduce binding of microorganisms having high cell wall lipid content.The hydrophobic surface may include a hydrophobic and positively chargedsurface. The hydrophobic and positively charged surface may include acoating comprising polydiallyldimethylammonium chloride. The hydrophobicsurface may include a hydrophobic and negatively charged surface. Thehydrophobic surface may include a surface modified by reaction withsilane reagents. The hydrophobic surface may include a surface modifiedby reaction with silane reagents including at least one of alkylsilanes, vinyl silanes, and amino silanes. The hydrophobic surface mayinclude a surface modified by reacting an amine-modified surface with apolymeric anhydride. The polymeric anhydride may be PA-18. The materialmay include at least one of a particulate, a bead, a membrane, a flowchannel, or a tube. The material may include a mineral oxide modified tohave a hydrophobic surface. The mineral oxide may be silica. Thematerial may include gold modified to have a hydrophobic surface. Thematerial may include at least one of a gold particulate, a gold bead, agold-coated particulate, or a gold-coated bead.

The material may further include a monolayer coating on the surfacecomprising a hydrophobic thiol compound.

A kit for use in isolating microorganisms having high cell wall lipidcontent from a specimen may be summarized as including a solid phasematerial having a modified surface with an affinity for microorganismsthat have a high cell wall lipid content; and instructions for use ofthe kit to isolate microorganisms having a high cell wall lipid content.The modified surface may include a hydrophobic surface, a hydrophobicand positively charged surface, or a hydrophobic and negatively chargedsurface. The hydrophobic and positively charged surface may include acoating comprising polydiallyldimethylammonium chloride. The modifiedsurface may include a surface modified by reaction with a silane, analkyl silane, a vinyl silane, or an amino silane. The modified surfacemay include a surface modified by reacting an amine-modified surfacewith a polymeric anhydride, including PA-18. The solid phase materialhaving a modified surface may include at least one of a particulate, abead, a membrane, a flow channel, or a tube. The solid phase materialhaving a modified surface may include at least one of a mineral oxide,silica, or gold.

A method of obtaining biological material of interest may be summarizedas including introducing a specimen containing a quantity of abiological material of interest into a chamber; and agitating thespecimen in the chamber with a medium that includes a mixed populationof particulate material and a fluid to mechanically lyse the specimenand to bind the biological material of interest to a particulate lysingmaterial and secondary biological materials to a particulate secondarybinding material; wherein the mixed population of particulate materialincludes the particulate lysing material and the particulate secondarybinding material, the particulate lysing material having an affinity forthe biological material of interest in the presence of the fluid, theparticulate secondary binding material having an affinity for secondarybiological materials other than the biological material of interest anda lateral dimension smaller than a lateral dimension of the particulatelysing material to allow selection of a mesh to selectivelysubstantially pass the particulate secondary binding material andsubstantially block passage of the particulate lysing material; andwherein the fluid induces binding of the biological material of interestto the particulate lysing material. Agitating the specimen with a mediumthat includes a mixed population of particulate material may includeagitating the specimen with a medium that includes the particulatelysing material and the particulate secondary binding material, eachincluding a plurality of beads. Agitating the specimen with a mediumthat includes a mixed population of particulate material may includeagitating the specimen with a medium that includes the particulatelysing material, the particulate lysing material including at least oneof a plurality of ceramic beads, a plurality of glass beads, a pluralityof zirconia (e.g., zirconium) beads, a plurality of silica beads, aplurality of sand, or a plurality of beads with a metal core coated by amaterial that facilitates binding of the biological material ofinterest. Agitating the specimen with a medium that includes a mixedpopulation of particulate material may include agitating the specimenwith a medium that includes the particulate secondary binding material,the particulate secondary binding material including at least one of aplurality of ceramic beads, a plurality of glass beads, a plurality ofzirconia (e.g., zirconium) beads, a plurality of silica beads, aplurality of sand, or a plurality of beads with a metal core coated by amaterial that facilitates binding of the secondary biological material.Obtaining a biological material of interest may include obtaining anucleic acid. Agitating the specimen with a medium that includes a mixedpopulation of particulate material to bind secondary biologicalmaterials may include agitating the specimen to bind biologicalmaterials that interfere with binding of the biological material ofinterest by the particulate lysing material. Agitating the specimen witha medium that includes a mixed population of particulate material tobind secondary biological materials may include agitating the specimento bind biological materials that interfere with subsequent reactionswith and analysis of the biological material of interest. Agitating thespecimen with a medium that includes a mixed population of particulatematerial to bind secondary biological materials may include agitatingthe specimen to bind lipids and lipid-like materials, proteins,polypeptides and polysaccharides. Agitating the specimen with a mediumthat includes a mixed population of particulate material may includeagitating the specimen with a medium that includes particulate secondarybinding material modified to include one or more agents to bind andremove from specimen lysate particular secondary biological materials.Agitating the specimen with a medium that includes a mixed population ofparticulate material may include agitating the specimen with a mediumthat includes particulate secondary binding material modified to includeone or more of Protein A, Protein G, or Protein L to bind and removeimmunoglobulins; anti-albumin antibody to bind and remove albumin;concanavalin A to bind and remove glycosylated proteins; or titaniumdioxide to bind and remove phosphorylated proteins. Agitating thespecimen with a medium that includes a mixed population of particulatematerial may include agitating the specimen with a medium that includesparamagnetic particulate secondary binding material. Agitating thespecimen with a medium that includes a mixed population of particulatematerial may include agitating the specimen with a medium that includesparticulate secondary binding material to bind and remove secondarybiological material before lysis of the specimen containing thebiological material of interest. Agitating the specimen with a mediumthat includes a mixed population of particulate material may includeagitating the specimen with a medium that includes particulate secondarybinding material to bind and remove secondary biological material forlater elution and analysis.

The method may further include providing a filter having passages sizedto substantially prevent passage of the particulate lysing material andsubstantially pass the particulate secondary binding material; andflowing a mixture of the agitated, lysed specimen with the fluid and themixed population of particulate material through the filter.

A kit for use in obtaining biological material of interest may besummarized as including a mixed population of particulate materialincluding a particulate lysing material and a particulate secondarybinding material, the particulate lysing material having affinity forthe biological material of interest, the particulate secondary bindingmaterial lacking affinity for the biological material of interest andhaving a size smaller than that of the particulate lysing material; andinstructions for use of the kit to obtain biological material ofinterest. Each of the particulate lysing material and the particulatesecondary binding material may include a plurality of beads. Theparticulate lysing material may include at least one of a plurality ofceramic beads, a plurality of glass beads, a plurality of zirconiabeads, a plurality of silica beads, a plurality of sand, or a pluralityof beads with a metal core coated by a material that facilitates bindingof the biological material of interest. The particulate secondarybinding material may include at least one of a plurality of ceramicbeads, a plurality of glass beads, a plurality of zirconia beads, aplurality of silica beads, a plurality of sand, or a plurality of beadswith a metal core coated by a material that facilitates binding of thesecondary biological material. The particulate secondary bindingmaterial may include one or more agents to bind and remove from specimenlysate particular secondary biological materials. The particularsecondary binding material may include one or more of Protein A, ProteinG, or Protein L to bind and remove immunoglobulins; anti-albuminantibody to bind and remove albumin; concanavalin A to bind and removeglycosylated proteins; or titanium dioxide to bind and removephosphorylated proteins. At least a portion of the particulate secondarybinding material may be paramagnetic.

The kit may further include a filter having passages sized tosubstantially prevent passage of the particulate lysing material andsubstantially pass the particulate secondary binding material.

A system to lyse specimens and isolate biological materials of interestmay be summarized as including a chamber to receive a specimencontaining the biological material of interest; a medium that includes amixed population of particulate material and a fluid; and an agitatorselectively operable to agitate the specimen in the chamber along withthe medium that includes the mixed population of particulate material;and wherein the mixed population of particulate material includes aparticulate lysing material and a particulate secondary bindingmaterial, the particulate lysing material having an affinity for thebiological material of interest in the presence of the fluid, theparticulate secondary binding material having an affinity for secondarybiological materials other than the biological material of interest anda lateral dimension smaller than a lateral dimension of the particulatelysing material to allow selection of a filter to selectivelysubstantially pass the particulate secondary binding material andsubstantially block passage of the particulate lysing material; andwherein the fluid induces binding of the biological material of interestto the particulate lysing material. Each of the particulate lysingmaterial and the particulate secondary binding material may include aplurality of beads. The particulate lysing material may include at leastone of a plurality of ceramic beads, a plurality of glass beads, aplurality of zirconia beads, a plurality of silica beads, a plurality ofsand, or a plurality of beads with a metal core coated by a materialthat facilitates binding of the biological material of interest. Theparticulate secondary binding material may include at least one of aplurality of ceramic beads, a plurality of glass beads, a plurality ofzirconia beads, a plurality of silica beads, a plurality of sand, or aplurality of beads with a metal core coated by a material thatfacilitates binding of the secondary biological material. Theparticulate secondary binding material may include at least one agent tobind and remove from specimen lysate particular secondary biologicalmaterials. The particulate secondary binding material may include atleast one of Protein A, Protein G, or Protein L to bind and removeimmunoglobulins; anti-albumin antibody to bind and remove albumin;concanavalin A to bind and remove glycosylated proteins; or titaniumdioxide to bind and remove phosphorylated proteins. At least a portionof the particulate secondary binding material may be paramagnetic.

The system may further include a filter having a mesh size to retain theparticulate lysing material and to pass the particulate secondarybinding material.

A method of capturing a biological material may be summarized asincluding introducing a fluid specimen containing the biologicalmaterial into a chamber of a device, the chamber containing a solidphase material that has an affinity for the biological material, thedevice having a fitting attached to the chamber, the fitting having athin filter insert that retains the solid phase material while allowingrapid flow of the fluid specimen through the filter insert; and flowingthe fluid specimen containing the biological material through theparticulate at low pressure and rapid rate, the fluid specimen exitingthe device via the filter insert. Introducing a fluid specimen into thechamber of a device may include introducing the specimen into a chambercontaining a solid phase material having an affinity for at least one ofa nucleic acid, a protein, a polypeptide, a His-tagged protein, aHis-tagged polypeptide, a lipid-containing biological material, aglycosylated protein, a phosphorylated protein, or a microorganismhaving a high cell wall lipid content. Introducing a fluid specimen intothe chamber of a device may include introducing the specimen into achamber containing a solid phase material having an affinity for morethan one biological material. Introducing a fluid specimen into thechamber of a device may include introducing the specimen into a chambercontaining a solid phase material comprising the solid phase material isa particulate or a bead. Introducing a fluid specimen into the chamberof a device may include introducing the specimen into a chambercontaining a solid phase material comprising a particulate or beadhaving a diameter or lateral dimension of at least 200 μm. Introducing afluid specimen into the chamber of a device having a fitting having afilter insert may include introducing the specimen into a device havinga filter insert of pore size between about 10 μm and about 200 μm andmore preferably between about 10 μm and about 200 μm and more preferablybetween about 40 μm and about 80 μm. Introducing a fluid specimen intothe chamber of a device having a fitting having a filter insert mayinclude introducing the specimen into a device having a filter insertwith a thickness between about 0.002″ and about 0.003″. Introducing afluid specimen into the chamber of a device having a fitting having afilter insert may include introducing the specimen into a device havinga wire mesh insert. Introducing a fluid specimen into the chamber of adevice having a fitting having a wire mesh insert may includeintroducing the specimen into a device having a stainless steel meshinsert.

The method may further include eluting the biological material capturedon the solid phase material by flowing an elution medium through thesolid phase at low pressure and uninhibited flow; and collecting aneffluent containing the biological material, the effluent exiting thedevice via the filter insert.

A device for capturing a biological material from a fluid specimen athigh flow rate and low operating pressure may be summarized as includinga chamber containing a solid phase material that has an affinity for thebiological material; and a fitting attached to the chamber, the fittinghaving a thin filter insert that retains the solid phase material whileallowing rapid flow of the fluid specimen at low pressure. The solidphase material may have an affinity for at least one of a nucleic acid,a protein, a polypeptide, a His-tagged protein, a His-taggedpolypeptide, a lipid-containing biological material, a glycosylatedprotein, a phosphorylated protein, or a microorganism having a high cellwall lipid content. The solid phase material may have an affinity formore than one biological material. The solid phase material may be aparticulate or a bead. The particulate or bead may have a diameter orlateral dimension of at least about 10 μm and more preferably about 100μm. The filter insert may have a pore size between about 10 μm and about200 μm and more preferably between about 40 μm and about 80 μm. Thefilter insert may have a thickness between about 0.002″ and about0.003″. The filter insert may be a wire mesh insert. The wire meshinsert may be stainless steel.

A method for removing fluid containing particulate matter from a chamberor a container may be summarized as including attaching a pipette tip toa pipette or a syringe, the pipette tip having a filter or mesh materialat least proximate an end of the pipette tip; inserting the end of thepipette tip having the filter or mesh material into the chamber orcontainer holding the fluid containing the particulate matter; andwithdrawing fluid from the chamber or container into the pipette tipthrough the filter or mesh. The filter or mesh material may be removablefrom or replaceable on the pipette tip.

A device for removing fluid containing particulate matter from a chamberor container may be summarized as including a pipette tip having afilter or mesh material at least proximate an end of the pipette tip.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

In the drawings, identical reference numbers identify similar elementsor acts. The sizes and relative positions of elements in the drawingsare not necessarily drawn to scale. For example, the shapes of variouselements and angles are not drawn to scale, and some of these elementsare arbitrarily enlarged and positioned to improve drawing legibility.Further, the particular shapes of the elements as drawn are not intendedto convey any information regarding the actual shape of the particularelements, and have been solely selected for ease of recognition in thedrawings.

FIG. 1A is a front elevational view of an apparatus to perform materiallysis, according to one illustrated embodiment.

FIG. 1B is a front, right side, top isometric view of the apparatus ofFIG. 1A.

FIG. 1C is a front, left side, bottom isometric view of the apparatus ofFIG. 1A.

FIG. 2A is a front elevational view of the apparatus of FIG. 1A with afront cover removed, according to one illustrated embodiment.

FIG. 2B is a front, right side, top isometric view of the apparatus ofFIG. 2A.

FIG. 2C is a front, right side, bottom isometric view of the apparatusof FIG. 2A.

FIG. 3 is a front, right side isometric view of a motor and drivemechanism of the apparatus of FIGS. 1A-2C.

FIG. 4 is a schematic view of a system to perform flow-throughprocessing, including an apparatus to perform material lysis, anupstream subsystem to provide material to be lysed, a downstreamsubsystem to analyze material that has been lysed, and a controlsubsystem, according to one illustrated embodiment.

FIG. 5 is a cross-sectional view of a container having a chamber thathouses material to be lysed, particulate lysing material, and materialthat has been lysed, according to one illustrated embodimentparticularly useful in flow-through lysing.

FIG. 6 is a flow diagram of a method of operating an apparatus, such asthe apparatus of FIGS. 1A-4, to perform lysing.

FIG. 7 is a flow diagram of a method of pumping material to be lysed ina flow-through lysing system such as that of FIG. 4 according to oneembodiment.

FIG. 8 is a flow diagram of a method of pumping material to be lysed ina flow-through lysing system such as that of FIG. 4, according toanother illustrated embodiment.

FIG. 9 is a flow diagram of a method of pumping material to be lysed ina flow through lysing system such as that of FIG. 4, according to yetanother illustrated embodiment.

FIG. 10 is a flow diagram of a method of pumping material to be lysed ina flow-through lysing system such as that of FIG. 4, according to stillanother illustrated embodiment.

FIG. 11 is a flow diagram of a method of evacuating lysed material in aflow-through lysing system such as that of FIG. 4, according to oneillustrated embodiment.

FIG. 12 is a flow diagram of a method of evacuating lysed material in aflow-through lysing system such as that of FIG. 4, according to anotherillustrated embodiment.

FIG. 13 is a method of pumping material to be lysed in a flow-throughlysing system such as that of FIG. 4, according to a further illustratedembodiment.

FIG. 14 is a flow diagram of a method of pumping material to be lysed ina flow-through lysing system such as that of FIG. 4, according to stilla further illustrated embodiment.

FIG. 15 is a method of operating a flow-through lysing system such asthat of FIG. 4 to analyze lysed material, according to one illustratedembodiment.

FIG. 16 is an exploded isometric view of a lysing apparatus according toanother illustrated embodiment.

FIG. 17 is a schematic diagram of a lysing system including a lysingapparatus, an upstream subsystem to provide material to be lysed, adownstream subsystem to analyze material that has been lysed, and acontrol subsystem, according to another illustrated embodiment.

FIG. 18 is a front elevation view of a lysing apparatus and pipetteaccording to one illustrated embodiment.

FIG. 19 shows a flow diagram of a method of operating a lysing apparatussuch as that of FIGS. 16 and 17, according to one illustratedembodiment.

FIG. 20 is a flow diagram of a method of evacuating material that hasbeen lysed from a chamber in operating a lysing apparatus such as thatof FIGS. 16 and 17, according to another illustrated embodiment.

FIG. 21 is a flow diagram of a method of receiving material to be lysedin a chamber in operating a lysing apparatus such as that of FIGS. 16and 17, according to one illustrated embodiment.

FIG. 22 is a flow diagram of a method of pumping material to be lysedinto a chamber in operating a lysing apparatus such as that of FIGS. 16and 17, according to one illustrated embodiment.

FIG. 23 is a flow diagram of a method of pumping material to be lysedinto a chamber in operating a lysing apparatus such as that of FIGS. 16and 17, according to another illustrated embodiment.

FIG. 24 is a flow diagram of a method of operating an impeller of alysing system such as that of FIG. 16, 17 or 18, according to oneillustrated embodiment.

FIG. 25 is a flow diagram of a method of operating an impeller of alysing system such as that of FIG. 16, 17 or 18, according to oneillustrated embodiment.

FIG. 26 is a flow diagram of a method of replacing a micromotor of alysing system such as that of FIG. 16, 17 or 18, according to oneillustrated embodiment.

FIG. 27 is a flow diagram of a method of operating a lysing apparatussuch as that of FIG. 18, according to one illustrated embodiment.

FIG. 28 is a flow diagram of a method of operating a lysing apparatussuch as that of FIG. 18, according to one illustrated embodiment.

FIG. 29 is a flow diagram of a method withdrawing lysed material from achamber of a lysing apparatus such as that of FIG. 18, according to oneillustrated embodiment.

FIG. 30 is a flow diagram of a method of reusing a micromotor of alysing apparatus such as that of FIG. 18, according to anotherillustrated embodiment.

FIG. 31 is a graph showing data representing an efficiency of lysis as afunction of lysing duration using an apparatus similar to that of FIG.4.

FIG. 32 is a graph showing a dependency of lysis efficiency on frequencyof oscillation.

FIG. 33 is a graph showing spore lysis as a function of lysis durationfor an apparatus similar to that of the embodiment of FIG. 16.

FIG. 34A is a schematic diagram of a bidirectional flow system forlysing, capture and elution including a sample and elution module, alysing module, a syringe pump, and a heater, according to oneillustrated embodiment.

FIG. 34B is a schematic diagram showing valve positions in the sampleand elution module during operation of the system in the embodiment ofFIG. 34A.

FIG. 35A is a plan view of a lysing apparatus having Luer-Lock couplers,according to one illustrated embodiment, and two syringes coupleable tothe lysing apparatus via the couplers.

FIG. 35B is an isometric view of the lysing apparatus of FIG. 35A.

FIG. 36 is a plan view of a plurality of lysing apparatus coupledsequentially to one another, according to one illustrated embodiment.

FIG. 37A is an isometric view of a manifold or array of lysingapparatus, according to one illustrated embodiment.

FIG. 37B is an isometric view of the manifold or array of lysingapparatus carried by a frame, according to one illustrated embodiment,the lysing apparatus positioned to deposit lysed material intorespective wells of a plate.

FIG. 38A is a side elevational view of a stopcock style lysing device,according to one illustrated embodiment, showing an inner portionrotated or configured to provide a first flow path via two selectedports.

FIG. 38B is a side elevational view of the stopcock style lysing deviceof FIG. 38A, showing the inner portion rotated or configured to providea second flow path via two selected ports.

FIG. 39A is an exploded isometric view of a stopcock style lysingdevice, according to one illustrated embodiment, showing an inner vesselhaving an open bottom portion, the inner vessel in a first orientationwith respect to an outer vessel.

FIG. 39B is an isometric view of a stopcock style lysing device of FIG.39A, showing an inner vessel received in an outer vessel, and an drivedevice including a motor and impeller received in the inner vessel.

FIG. 39C is an isometric view of the inner vessel of FIG. 39A, showingthe inner vessel in a second orientation, different from the orientationillustrated in FIG. 39A.

FIG. 40A is an exploded side elevational view of a stopcock style lysingdevice, according to another illustrated embodiment, showing an innervessel with a closed bottom portion.

FIG. 40B is an bottom plan view of a stopcock style lysing device ofFIG. 40A.

FIG. 41 is a flow diagram of aspects of a method of processing a nucleicacid-containing specimen including washing particle-bound nucleic acid,according to one embodiment.

FIG. 42 is a flow diagram of an aspect of a method of preparing anucleic acid-containing specimen for processing by a method such as thatof FIG. 41, according to one embodiment.

FIG. 43 is a flow diagram of an aspect of a method of preparing anucleic acid-containing specimen for processing by a method such as thatof FIG. 41, according to another embodiment.

FIG. 44 is a flow diagram of an aspect of a method of preparing anucleic acid-containing specimen for processing by a method such as thatof FIG. 41, according to yet another embodiment.

FIG. 45 is a flow diagram of an aspect of a method of processing anucleic acid-containing specimen by a method such as that of FIG. 41including eluting particle-bound nucleic acid, according to oneembodiment.

FIG. 46 is a flow diagram of an aspect of a method of processing anucleic acid-containing specimen by a method such as that of FIG. 41including providing eluted nucleic acid for amplification, according toone embodiment.

FIG. 47 is a flow diagram of aspects of a method of processing aphosphate-containing polyanion using an amine-containing solid phasematerial, according to one embodiment.

FIG. 48 is a flow diagram of an aspect of a method of processing aphosphate-containing polyanion specimen by a method such as that of FIG.47 including eluting particle-bound phosphate-containing polyanion,according to one embodiment.

FIG. 49 is a flow diagram of aspects of a method of processing asulfonate-containing polyanion using an amine-containing solid phasematerial, according to one embodiment.

FIG. 50 is a flow diagram of an aspect of a method of processing asulfonate-containing polyanion specimen by a method such as that of FIG.49 including eluting particle-bound sulfonate-containing polyanion,according to one embodiment.

FIG. 51 is a flow diagram of a method of capturing a microorganismhaving a high content of cell wall lipid by a hydrophobic surface of amaterial, according to one embodiment.

FIG. 52 is a flow diagram of a method of lysing a microorganism having ahigh cell wall lipid content captured by a hydrophobic surface of amaterial, according to one embodiment.

FIG. 53 is a flow diagram of a method of amplifying nucleic acid from alysate of a microorganism having high cell wall lipid content capture bya hydrophobic surface of a material, according to one embodiment.

FIG. 54 is a flow diagram of a method of capturing a microorganismhaving a high content of cell wall lipid by a hydrophobic and positivelycharged surface of a material, according to one embodiment.

FIG. 55 is a flow diagram of a method of lysing a microorganism having ahigh cell wall lipid content captured by a hydrophobic and positivelycharged surface of a material, according to one embodiment.

FIG. 56 is a flow diagram of a method of amplifying nucleic acid from alysate of a microorganism having high cell wall lipid content capture bya hydrophobic and positively charged surface of a material, according toone embodiment.

FIG. 57 is a flow diagram of a method of capturing a microorganismhaving a high content of cell wall lipid by a hydrophobic and negativelycharged surface of a material, according to one embodiment.

FIG. 58 is a flow diagram of a method of lysing a microorganism having ahigh cell wall lipid content captured by a hydrophobic and negativelycharged surface of a material, according to one embodiment

FIG. 59 is a flow diagram of a method of amplifying nucleic acid from alysate of a microorganism having high cell wall lipid content capture bya hydrophobic and negatively charged surface of a material, according toone embodiment.

FIG. 60 is a flow diagram of a method of processing a biologicalmaterial with a mixed population of particulate materials of differentsizes, according to one embodiment.

FIG. 61 is a flow diagram of a method of processing a biologicalmaterial with a mixed population of particulate materials of differentsizes, according to another embodiment.

FIG. 62 is a flow diagram of a method of processing a biologicalmaterial with a mixed population of particulate materials of differentsizes using a filter to separate particles by size, according to oneembodiment.

FIG. 63 is a flow diagram of a method of processing a biologicalmaterial with a mixed population of particulate materials of differentsizes using a filter to separate particles by size, according to anotherembodiment.

FIG. 64 is a flow diagram of aspects of a method of processing abiological material on a solid phase at high flow and low pressure,according to one embodiment.

FIG. 65 is a flow diagram of further aspects of a method of processing abiological material on a solid phase at high flow and low pressure as inFIG. 56, according to another embodiment.

FIG. 66 is a flow diagram of aspects of a method of processing a fluidcontaining a particulate material, according to one embodiment.

FIG. 67A is a pipette tip device for processing a fluid containing aparticulate material, according to one illustrated embodiment.

FIG. 67B is a pipette tip device for processing a fluid containing aparticulate material, according to another illustrated embodiment.

FIG. 68 is an exploded isometric view of a device for capturing abiological material from a fluid specimen according to one illustratedembodiment.

FIG. 69 is a chart showing several silica beads modified with differentchemical moieties which can be employed in many different embodiments.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various disclosedembodiments. However, one skilled in the relevant art will recognizethat embodiments may be practiced without one or more of these specificdetails, or with other methods, components, materials, etc. In otherinstances, well-known structures associated with micromotors,controllers including motor controllers, and control systems such asprogrammed general purpose computing systems and the like have not beenshown or described in detail to avoid unnecessarily obscuringdescriptions of the embodiments. In other instances, methods commonlyknown for use with and manipulation of nucleic acids, proteins,polypeptides, and other biological materials have not been described asthey would be readily available to those of ordinary skill in the art ofsuch materials. Such common methods include, for example, PCR and heatdenaturation of DNA.

Unless the context requires otherwise, throughout the specification andclaims which follow, the word “comprise” and variations thereof, suchas, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is, as “including, but not limited to.”

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment. Thus, the appearances of the phrases “in one embodiment” or“in an embodiment” in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. It should also be noted that the term “or”is generally employed in its sense including “and/or” unless the contentclearly dictates otherwise.

The headings and Abstract of the Disclosure provided herein are forconvenience only and do not interpret the scope or meaning of theembodiments.

A number of embodiments of lysis apparatus, systems and methods of useare described herein. The lysis apparatus and systems perform lysis on amaterial to be lysed using lysing particulate material, to produce lysedmaterial or material that has been lysed. The material to be lysed maytake the form of biological materials, for example cells, spores,tissue, yeast, fungi, plants, bacteria, etc., typically suspended in aliquid medium. The lysing particulate material may take a variety offorms. While often referred to herein as beads, the term bead is notmeant to be limiting with respect to size or shape. The beads may, forexample, comprise ceramic, glass, zirconia, zirconia/silica, zirconiumsilicate, metal, plastic, nickel, tungsten, tungsten carbide. Yttriumstabilized zirconia, sand, and/or particles of any geometry such asshard or of random shape. The lysed material may likewise take a varietyof forms, for example nucleic acids, polypeptides, proteins,organelles-nuclei, mitochondria, lysosomes, chloroplasts, endoplasmicreticulum, etc.

Various embodiments of the material separation and/or lysis apparatusand systems may, for example, operate in: 1) a batch mode, 2)flow-through stop or semi-batch mode, or 3) continuous flow-throughmode. In batch mode, a container having a chamber holding a sample ofmaterial to be lysed is located in a holder and oscillated. Thecontainer is removed after sufficient oscillation and the lysed materialis recovered. In the flow-through stop or semi-batch mode, a sample ofmaterial to be lysed flows into to fill the chamber. The container isthen oscillated until sufficiently lysed. The chamber is evacuated ofthe lysed material. In the flow-through mode, a sample of material to belysed flows through the chamber of the container during oscillation at adesired flow rate, providing a desired or defined residence time withinthe chamber. In the flow-through stop or semi-batch mode, the sample maybe abutted by an immiscible liquid or gas and the chamber may beevacuated by a blast of a fluid, for example a liquid or a gas.

At least some of the embodiments take advantage of the understandingthat the forces responsible for mechanical rupture of biological samplesscale with the oscillation frequency squared, and that by employingrelatively small sample sizes, the various embodiments described hereincan achieve relatively higher frequencies than commercially availableapparatus, resulting in rapid and efficient lysis.

A number of embodiments of systems and methods for extraction, captureand elution of biological materials, in particular nucleic acids such asDNA, are described herein. Biological specimens, such as cells orviruses, may be lysed by mechanical disruption in a lysing chambercontaining particulate material, for example beads made from silicaand/or zirconia. The volume of the lysing chamber may be crowded withthe particulate material. The particulate material in the lysing chambermay be driven rapidly by an impeller connected to a small motor to lysethe biological specimens. The motor may be disposable. Alternatively,the lysing chamber may be oscillated to drive the particulate materialto lyse the biological specimens. Further, treatment of the contents ofthe lysing chamber may include ultrasonic treatment. Such differenttypes of mechanical disruption allow lysis to occur without the use ofharsh chemicals, such as chaotropic agents. In comparison to standardprocedures for preparation of biological materials, the proceduresdisclosed herein may save time by eliminating wash steps that aretypically included to remove harsh chemicals. Chemical conditions withinthe lysing chamber may be controlled during lysis to allowsimultaneously lysis of the biological specimen and binding orcollection of the biological material released by lysis, e.g., DNA orprotein or both, on the particulate material. The apparatus or systemmay then be operated to alter chemical and/or flow conditions within thelysing chamber to elute the biological material of interest from theparticulate material. The systems and methods disclosed herein thusadvantageously allow simple, efficient approaches to lysis, capture andelution of biological materials from biological specimens. Thesurprisingly advantageous approaches involve appropriately timed, simplecontrol of flow direction and chemical compositions of fluids within alysing system. The biological material, e.g., DNA, may then be subjectedto testing or analysis or used for other purposes. The absence of harshreagents during lysis may not only save time but also yield materialsthat are more suitable for use in subsequent procedures. Thus, thedisclosed systems and methods provide rapid and efficient lysis ofspecimens, e.g., cells, and capture of biological materials, e.g., DNA,in a single chamber by sequential use of fluids having chemicalcompositions particularly appropriate for lysis, capture and elution.Various specific embodiments will now be discussed.

FIGS. 1A-1C and 2A-2C show an apparatus 10 operable to perform lysing ona material to be lysed contained in a container 12, according to oneillustrated embodiment. In some embodiments, off-the-shelf vials andtubes may be employed as the container 12 to hold specimens of materialto be lysed and the lysing particulate material or other material, forexample PCR or Eppendorf tubes. While illustrated in FIGS. 1A-1C and2A-2C in a batch mode, the lysis apparatus 10 may be used in aflow-through stop or semi-batch mode or in a continuous mode asillustrated in FIG. 4.

The container 12 may be removably coupled to an arm 14 via a holder 16.The holder 16 may take a variety of forms. For example, the holder 16may take the form of a U-shaped clamp or other member. The holder 16 mayinclude a fastener (e.g., screw, bolt, etc.) 16 a operable to secure theholder 16 in a container securing configuration. Alternatively, theholder 16 may be resilient and biased into the container securingconfiguration.

The arm 14 may be coupled to pivot about an axle 18 such that thecontainer 12 oscillates along an arcuate path 20. Oscillation along anarcuate path 20 achieves confined periodic flow fields with angularaccelerations that provide strong particulate flow fields and largeshear rates between beads in a liquid solution or slurry. Experiments bythe applicants have demonstrated that miniaturized geometries canprovide superior lysis through the application of high frequencies(e.g., greater than approximately 100 Hz). Since the relative forces onnon-neutral density beads in a liquid scale according to ω²r, where ωrepresents angular velocity and r is the distance of a bead from thecenter of rotation, a small increase in angular speed can allow for asubstantial decrease in size to attain similar performance. Linearoscillatory motions, even at high frequencies result in little lysis ofbiological samples, while those with an arc motion may achieve lysisthat is superior to commercially available bead-based lysis apparatus.High-speed movies clearly show that linear motions result in periodicconcentration of beads followed by expansion of beads away from oneanother, but relatively little relative motion of beads that is notalong the axis of motion. In contrast, where a container oscillates inan arc, the beads are seen to compress to higher density just as astrong swirl is induced, resulting in very effective lysing. Collisionsand shearing provided by the relative motion of the suspended beadscontribute to the high efficiency of the lysing.

The arm 14 may be a rigid arm, i.e., an arm that does not appreciablybend during oscillation with a load having a mass at least roughlyequivalent to an expected load of a container containing a material tobe lysed and a lysing particulate material. Alternatively, the arm 14may be a flexible arm, i.e., an arm that does appreciably bend duringoscillation with a load having a mass at least roughly equivalent to anexpected load of a container containing a material to be lysed andoptionally a lysing particulate material.

As best illustrated in FIGS. 2A-2C and 3 in which a cover plate 24 isremoved, the arm 14 may be driven via a motor 22 and a drive mechanism26, which may take the form of a four-bar linkage. In particular, ashaft 28 of the motor 22 drives a first member such as a bar, here inthe form of eccentric cam 30. The eccentric cam 30 is received in a bore32 of a second member or connecting arm 34. The connecting arm 34 isdrivingly coupled to the holder 16 by the axle 18 of a rocker arm 36.The drive mechanism 26 provides a low cost, reliable mechanism torealize relatively high frequency oscillatory motion along the arcuatepath 20. While such frequencies may not be considered high for othertypes of devices, of instance rotating devices or ultra-sonic devices,such frequencies are considered high oscillating type devices.

FIG. 4 shows a flow-through lysis system 400 according to oneillustrated embodiment. As described in more detail herein, theflow-through lysis system 400 may be operated in a flow-through stop orsemi-batch mode, or in a continuous flow mode.

The flow-through system 400 includes a lysing apparatus 410 and acontainer 412, which may be similar to those described in previousembodiments. For example, the lysing apparatus 410 may include an arm414 and holder 416 to hold the container 412 as the container pivotallyoscillates about an axle 418.

The flow-through lysis system 400 may include an upstream subsystem 438to deliver material to be lysed. For example, the upstream subsystem 438may include a pump 440 operable to pump or otherwise deliver material tobe lysed to the container 412. The upstream subsystem 438 may alsoinclude a reservoir 442 that holds the material to be lysed.

The upstream subsystem 438 may additionally or alternatively include amechanism to collect material to be lysed, for example a samplingapparatus 439. The sampling apparatus 439 may be manually operated ormay be automatic. The sampling apparatus 439 may, for example, samplethe ambient environment, for example the air or atmosphere, water orfluids, soil or other solids. The sampling apparatus 439 may include avacuum or mechanism to create a negative pressure to extract a sample.The sampling apparatus 439 may include an actuator, for example an armwith a shovel or broom to retrieve samples. The sampling apparatus mayinclude an actuator, for example a needle and syringe to examplesamples.

The material to be lysed may be delivered via one or more conduits, forexample, a tube 444 a to an entrance 446 a of the container 412. Thetube 444 a may be reinforced at one or both ends, for example, beingreinforced with multiple layers of concentrically arranged tubes 448 a.The tube 444 a may have a length L₁ that is sufficiently long to allowthe container 412 and arm 414 to oscillate, while being sufficientlyshort as to prevent resonance in the tube. The length L₁ would be afunction of the density, the rigidity, or the attachment method of thetube 444 a as well as the density, mass and/or rigidity of any materialto be lysed carried therein.

The flow-through lysis system 400 may further include a downstreamanalysis subsystem 449. The downstream analysis subsystem 449 mayinclude one or more downstream analysis apparatus 450. The downstreamanalysis apparatus 450 may take any of a variety of forms. For example,the downstream analysis apparatus 450 may include a nucleic acidamplification instrument, electron-microscope, western blottingapparatus, mass spectrometer, gas chromatograph, etc.

The downstream analysis subsystem 449 may further include one or morecomputing systems 452 communicatively coupled to the downstream analysisapparatus 450. The computing system 452 may be coupled to one or morenetworks 453, for example a local area network (LAN), a wide areanetwork (WAN) such as the Internet, and/or a wireless wide area network(WWAN). The computer system 452 may provide information about theresults of an analysis performed on lysed material via the network 453.For example, the computing system 452 may automatically provide an alertor other message to suitable system based on the results of theanalysis. Such may, for example, be used to provide an alert when atoxic or dangerous substance or condition is detected.

The downstream analysis apparatus 450 may be fluidly communicativelycoupled to an exit 446 b of the container 412 via one or more conduits,for example, tube 444 b. The tube 444 b may be reinforced at one or bothends, for example, by one or more concentrically arranged lengths oftube 448 b. The tube 444 b may have a length L₂ that is sufficientlylong as to allow the container 412 and arm 414 to oscillate freely whilebeing sufficiently short as to prevent resonance of the tube 444 b. Thelength L₂ may be based on the density, the rigidity, or the attachmentmethod of the tube 444 b as well as a density, mass and/or rigidity ofany material carried therein.

The flow-through lysis system 400 may further include one or morecontrol systems 454. The control system 454 may take the form of one ormore motor controllers and/or computing systems. The control system 454may be configured to operate the flow-through system 400 in aflow-through stop or semi-batch mode and/or in a flow-through continuousflow mode. The control systems 454 may, for example, be communicativelycoupled to control the lysing apparatus 410 and/or pump 440.

The flow-through system 400 provides a number of advantages over batchbased apparatus. For example, some types of beads may have an affinityfor certain bio-products that are released on lysis, so some of the cellcontents may be “lost” due to adsorption on the bead surfaces. Theflow-through design may advantageously automatically elute the adsorbedbiomolecules. It also avoids difficult or additional acts that may berequired in batch mode configurations to evacuate the chamber. Forexample, the flow-through embodiments may eliminate any possible need toblast the chamber with a fluid such as air to clear the chamber of thelysed material.

FIG. 5 shows a container 512 according to one illustrated embodiment.

The container 512 may have an entrance 546 a to provide fluidcommunication from an exterior 560 of the container to a chamber 562 ofthe container 512. The container 512 may include an exit 546 b providingfluid communication between the exterior 560 and the chamber 562 of thecontainer 512. A first tube 544 a may be coupled to the container 512 toprovide material to be lysed 564 to the chamber 562 via the entrance 546a. As noted previously, the tube 544 a may be reinforced, for example,with one or more layers of concentrically arranged tubing 548 a. Asecond tube 544 b may be coupled to the container 512 via the exit 546 bto remove lysed material 566 via the exit 546 b. In some embodiments,the container 512 may include attachment structures to attach orotherwise couple or secure the tubes 544 a, 544 b. For example, thecontainer 512 may include a ribbed nipple 568 a at the entrance 546 aand/or a ribbed nipple 568 b at or proximate the exit 546 b.

The container includes lysing material 570. The lysing material 570 maytake a variety of forms, for example, a plurality of beads. The beadsmay take a variety of forms including one or more of ceramic beads,glass beads, zirconia beads, zirconia/silica beads, metal beads, plasticbeads, sand, and/or metal beads that are coated with a bio-affinitymaterial or receptor, such as a sequence specific probe or an antibody.The beads may have a variety of diameters, for example, betweenapproximately 10 microns and approximately 600 microns.

In the flow through embodiments, the container 512 may include a firstfilter 572 a positioned relatively proximate the entrance 546 a and asecond filter 572 b positioned relatively proximate the exit 546 b. Thefirst and second filters 572 a, 572 b form a particulate retainment area574 in which the lysing particulate material 570 is retained. Inparticular, the filters 572 a, 572 b may have a plurality of openingssized to substantially pass the material to be lysed 564 and the lysedmaterial 566, respectively, while blocking the particulate lysingmaterial 570. The container 512 may include one or more structures, forexample, tabs or annular ridges 576 a, 576 b to retain the first andsecond filters 572 a, 572 b in place. Filters may, for example take theform of nylon or stainless steel mesh filter.

Many of the embodiments include chambers which have multiple openingsand which include filters of mesh to retain particulates in the chamber.In some embodiments it is advantageous to use filters having differentpore sizes for different chamber openings. For example, a filter at theopening where sample enters the chamber may have a larger pore size tofacilitate the entry of cells or particles of tissue while a filter atthe opening which serves as an exit to the chamber may have a filterwith a smaller pore size to retain the cells or particles of tissuewithin the chamber. This embodiment would, for example, retain largecells in the chamber of a lysis device until the cells are effectivelylysed.

The embodiments of FIGS. 1A-5 may advantageously allow extremely highpacking densities. In these embodiments, the volume of particulatematerial may advantageously exceed the volume of material to be lysed ormay exceed the volume of material that has been lysed. Additionally oralternatively, these embodiments may advantageously have essentially noair in the chamber. As used herein, essentially no air means that thechamber is free of air other than small bubbles which may beunintentionally entrapped in the chamber. Such may increase lysingefficiency and prevent undesirable heating of the system from frictionassociated with liquid-air contact line motions.

FIG. 6 shows a method 600 of operating an apparatus such as thatillustrated in FIGS. 1A-4 to lyse material, according to one illustratedembodiment.

At 602, material to be lysed is received in the chamber of thecontainer. The chamber may already hold lysing particulate material. At604, the container is oscillated along an arcuate path. The oscillationproduces large variations in movement between respective ones of thelysing particulate material. Such variations are more pronounced than intranslational or rotational movements. At 606, the lysed material isremoved from the chamber of the container.

FIG. 7 shows a method 700 of pumping material to be lysed in aflow-through lysing system such as the one of FIG. 4, according to oneillustrated embodiment.

At 702, the material to be lysed is pumped into the chamber of thecontainer.

FIG. 8 shows a method 800 of pumping material to be lysed in aflow-through lysing system such as that of FIG. 4, according to oneillustrated embodiment.

At 802, the material to be lysed is intermittently pumped into thechamber of the container while the container is oscillated. Such issuitable for the flow-through stop or semi-batch mode.

FIG. 9 shows a method 900 of pumping material to be lysed in aflow-through lysing system such as that of FIG. 4, according to anotherillustrated embodiment.

At 902, the material to be lysed is intermittently pumped into thechamber such that the material to be lysed spends a sufficient time inthe chamber to achieve a desired level of lysing. Thus, if it isdetermined that 30 seconds of oscillation achieves a desired level oflysing, the pump may be intermittently operated to load the chamber withmaterial to be lysed approximately every 30 seconds. Oscillation timesof few seconds or tenths of seconds may be suitable. Such operation issuitable for the flow-through stop or semi-batch mode.

FIG. 10 shows a method 1000 of pumping material to be lysed in aflow-through lysing system such as that of FIG. 4, according to anotherillustrated embodiment.

At 1002, the material to be lysed is intermittently pumped into thechamber such that the chamber is completely evacuated of the lysedmaterial during each cycle of the intermittent pumping. Such is suitablefor the flow-through stop or semi-batch mode.

FIG. 11 shows a method 1100 of evacuating lysed material in aflow-through lysing system such as that of FIG. 4, according to anotherillustrated embodiment.

At 1102, the chamber is evacuated of the lysed material during eachcycle of the intermittent pumping by pumping into the chamber morematerial to be lysed. Such is suitable for the flow-through stop orsemi-batch mode.

FIG. 12 shows a method 1200 of operating a lysing apparatus such as thatof FIG. 4, according to another illustrated embodiment.

At 1202, the chamber is evacuated of the lysed material each cycle ofthe intermittent pumping by pumping an inert fluid into the chamber. Theinert fluid may take the form of a liquid or gas, and may be immisciblewith the lysed material or material to be lysed. Such is suitable forthe flow-through stop or semi-batch mode.

FIG. 13 shows a method 1300 of operating a continuous lysing apparatus,according to one illustrated embodiment.

At 1302, the material to be lysed is continuously pumped into thechamber of the container while the container is oscillated. Such issuitable for the flow-through continuous mode.

FIG. 14 shows a method 1400 of operating a flow-through lysingapparatus, according to another illustrated embodiment.

At 1402, a flow rate of the pumping of the material to be lysed isadjusted based at least in part on the length and free volume of thechamber such that the material to be lysed spends sufficient time in thechamber (i.e., desired or defined residence time) to achieve a desiredlevel of lysing. Such is suitable for the flow-through continuous mode.

FIG. 15 shows a method 1500 of operating a flow-through lysingapparatus, such as that of FIG. 4, according to another illustratedembodiment.

At 1502, the lysed material removed from the chamber of the container isdirected to at least one analysis device. At 1504, the lysed material isanalyzed. Analysis may take a variety of forms, for example analysiswith electron-microscope, western blotting, mass spectrometry, gaschromatography, etc. Such is suitable for any of the modes, andparticularly suited to the flow-through modes.

FIG. 16 shows a flow-through lysing apparatus 1600 according to anotherillustrated embodiment. As described in more detail herein, the flowthrough lysis system 1600 may be operated in a flow-through stop orsemi-batch mode, or in a continuous flow mode.

The flow-through lysing apparatus 1600 includes a container 1602 havinga chamber 1604, and a micromotor 1606 coupled to drive an impeller 1608.

As illustrated, the chamber 1604 may have a first opening 1604 a thatserves as an entrance providing fluid communication from an exterior1610 of the container 1602 to the chamber 1604. Also as illustrated, thechamber 1604 may have a second opening 1604 b that serves as an exit,providing fluid communication from the chamber 1604 to the exterior1610. The container 1602 may further have a third opening 1604 c sizedto receive the impeller 1608 and to sealingly engage an outer portion ofthe micromotor 1606. Some embodiments may include a bushing or O-ring toform or enhance the sealing between the micromotor 1606 and thirdopening 1604 c.

A first coupler 1610 a may include a stem 1612 a sized to be sealinglyreceived in the opening 1604 a to provide fluid communication into thechamber 1604. The stem 1612 a may be threaded with the hole 1604 ahaving a complementary thread. The first coupler 1610 a may include anattachment structure, for example, a ribbed nipple 1614 a to secure atube 1616 a and provide a flow of material to be lysed to the chamber1604. An O-ring 1618 a, or other similar structure, may enhance a sealbetween a flange of the first coupler 1610 a and the container 1602.

A second coupler 1610 b may include a stem 1612 b sized to be sealinglyreceived in the opening 1604 b to provide fluid communication into thechamber 1604. The stem 1612 b may be threaded with the hole 1604 bhaving a complementary thread. The second coupler 1610 b may include anattachment structure, for example, a ribbed nipple 1614 b to secure atube 1616 b and provide a flow of material to be lysed to the chamber1604. An O-ring 1618 b, or other similar structure, may enhance a sealbetween a flange of the second coupler 1610 b and the container 1602.

Filters 1619 a, 1619 b may be positioned in the chamber to retain lysingparticulate material therebetween. The filters 1619 a, 1619 b may, forexample, take the form of nylon mesh filters with 50 micron openingsmounted to suitable fittings.

The micromotor 1606 may, for example, take the form of a micromotorhaving a 4 mm diameter, and may be capable of driving the impeller athigh speed, for example approximately 50,000 RPM, when not in thepresence of liquid and beads. The impeller 1608 may be a nylon oracrylic impeller having a number of vanes. The vanes may be straight,without curvature or angle of attachment, such that movement of materialis primarily circumferential. Should axial/horizontal movement of thematerial through the chamber be desirable, for example in a flow-throughmode (e.g., FIGS. 16 and 17), such axial or flow movement comes frompumping and not from rotation of the impeller. This allows more precisecontrol over amount of time that the material remains in the chamber andhence is subject to lysis. The vanes may, for example, produce aperiodic flow at a frequency nearly 5 times as high as the embodimentsof FIGS. 1A-4, however with a smaller amplitude of motion.

The lysing apparatus 1600 may also include a controller 1620 coupled tocontrol the micromotor 1606. The controller 1620 may, for exampleinclude a motor controller and/or a programmed general purpose computingsystem, a special purpose computer, an application specific integratedcircuit (ASIC) and/or field programmable gate array (FPGA). Thecontroller 1620 may for example, be programmed or configured to causethe motor to pulsate. Pulsating may increase the effectiveness of thelysing.

FIG. 17 shows a flow-through lysing system 1700 according to oneillustrated embodiment. As described in more detail herein, theflow-through lysis system 1700 may be operated in a flow-through stop orsemi-batch mode, or in a continuous flow mode.

The flow-through lysing system 1700 includes a container 1702 having achamber (not illustrated in FIG. 17), openings 1704 a, 1704 c (only twoillustrated), and a micromotor 1706 coupled to an impeller (not shown inFIG. 17). The opening or entrance 1704 may be fluidly communicativelycoupled to a pump 1720 that delivers material to be lysed from areservoir 1722 via a first conduit or tube 1716 a. A second opening orexit may deliver lysed material to one or more downstream analysisapparatus 1724 via one or more conduits such as tubes 1716 b. Aspreviously noted, downstream analysis may take a variety of forms, forinstance nucleic acid amplification, electrophoresis, western blotting,mass spectrometry, gas chromatography, etc. The downstream analysisapparatus 1724 may be communicatively coupled to one or more computingsystems 1726. The flow-through lysing system 1700 may also include oneor more control systems 1728 which may control the micromotor 1706and/or pump 1720. The control system 1728 may for example synchronizethe pumping and oscillation, for example to implement a flow-throughstop or semi-batch mode. The control system 1728 may for example controlthe pumping to attain a desired or defined residence time of thematerial in the chamber to achieve a desired or defined level of lysing,for example to implement a flow-through continuous mode.

The embodiments of FIGS. 16 and 17 may advantageously allow extremelyhigh packing densities. In these embodiments, the volume of particulatematerial may advantageously exceed the volume of material to be lysed ormay exceed the volume of material that has been lysed. Additionally oralternatively, these embodiments may advantageously have essentially noair in the chamber. As used herein, essentially no air means that thechamber is free of air other than small bubbles which may beunintentionally entrapped in the chamber. Such may increase lysingefficiency and prevent undesirable heating of the system from frictionassociated with liquid-air contact line motions.

FIG. 18 shows a lysing system 1800 according to another illustratedembodiment. The lysing system 1800 is particularly suitable for batchmode lysing operations.

The lysing system 1800 includes a container 1802 having a chamber 1804that has a single opening 1804 a to provide fluid communication with anexterior of the container 1802. The apparatus 1800 includes a micromotor1806 coupled to drive an impeller 1808 that is received in the chamber1804. A portion of the micromotor 1806 is sized to form a sealingengagement with the container 1802 to seal the opening 1804 a. Someembodiments may include one or more bushings or O-rings (not shown) toensure the seal.

Initially, the chamber 1804 is packed with material to be lysed 1810 andlysing particulate material 1812. After rotation of the impeller 1808,for a sufficient length of time, the chamber 1804 contains material thathas been lysed and the lysing particulate material 1812. The micromotor1806 and impeller 1808 may then be removed and the lysed material may beextracted, for example using a pipette 1814. The chamber 1804 of thebatch mode embodiments may not be as densely packed as in flow-throughembodiments since room may be required for the apparatus to withdraw thelysed material.

In some embodiments, off-the-shelf vials and tubes may be employed asthe container 1802 to hold specimens of material to be lysed and thelysing particulate material, for example PCR or Eppendorf tubes.

The embodiment of FIG. 18 may advantageously allow extremely highpacking densities. In these embodiments, the volume of particulatematerial may advantageously exceed the volume of material to be lysed ormay exceed the volume of material that has been lysed. This embodimentis less likely to ensure that there is essentially no air in the chambersince room may be required for receiving the withdrawal apparatus (e.g.,pipette). However, where possible, elimination of air in the chamber mayincrease lysing efficiency and prevent undesirable heating of the systemfrom friction associated with liquid-air contact line motions.

FIG. 19 shows a method 1900 of operating a flow-through lysing apparatusand/or system according to one illustrated embodiment. Such may beuseful in a flow-through stop or semi-batch mode or in a flow-throughcontinuous mode.

At 1902, material to be lysed is received in the chamber of a containervia an entrance. The chamber may already hold lysing particulatematerial. At 1904, the micromotor drives the impeller to cause thelysing particulate material to lyse the material to be lysed. At 1906,material that has been lysed is expelled from the chamber of thecontainer via an exit.

FIG. 20 shows a method 2000 of evacuating material that has been lysedfrom a chamber, according to one illustrated embodiment.

At 2002, the material that has been lysed may be expelled via a firstfilter position before the exit in a flow path of material through theapparatus or system.

FIG. 21 shows a method 2100 of receiving material to be lysed in achamber, according to another illustrated embodiment.

At 2102, the material to be lysed is received in the chamber via asecond filter positioned following the entrance of the chamber in theflow path through the apparatus or system.

FIG. 22 shows a method 2200 of pumping material to be lysed into achamber, according to another illustrated embodiment.

At 2202, the material to be lysed is intermittently pumped into thechamber via the entrance. Such may be particularly suitable forflow-through stop or semi-batch mode operation.

FIG. 23 shows a method 2300 of pumping material to be lysed into achamber, according to one illustrated embodiment.

At 2302, the material to be lysed is continuously pumped into thechamber of the container via the entrance, at a flow rate that providesfor a resident time of the material to be lysed in the chamber that issufficiently long to achieve a desired or defined level of lysing. Themicromotor may continuously drive the impeller to lyse the material.Such may be particularly suitable for flow-through continuous modeoperation.

FIG. 24 shows a method 2400 of operating an impeller of a lysing system,according to one illustrated embodiment.

At 2402, the micromotor pulsatingly drives the impeller. Pulsations maybe achieved by varying a voltage or current delivered to the micromotor.Pulsating may achieve a higher efficiency of lysing, thereby increasingthroughput or decreasing time required to achieve a desired or definedlevel of lysing.

FIG. 25 shows a method 2500 of operating an impeller of a lysing systemaccording to one illustrated embodiment.

At 2502, the micromotor drives the impeller at greater than 10,000 RPMin the presence of liquid and beads. Driving the impeller at arelatively high speed achieves a desired or defined level of lysing.

FIG. 26 shows a method 2600 of replacing a micromotor of a lysing systemaccording to one illustrated embodiment.

At 2602, the micromotor may be replaced with a new micromotor. At 2604,the old micromotor may be disposed or recycled. This may be particularlyuseful since it is difficult to seal the internal elements (e.g., rotor,stator) of the high speed micromotor from exposure to the ambientenvironment, thus the micromotors may fail more frequently than in otherembodiments or environments.

FIG. 27 shows a method 2700 of operating a batch based lysing apparatusaccording to one illustrated embodiment. The method 2700 may beparticularly useful for use with the embodiment of FIG. 18.

At 2702, material to be lysed is received in a chamber of a firstcontainer via an entrance. The chamber may already hold a lysingparticulate material or the lysing material may be provided into thechamber with or after the material to be lysed.

At 2704, an impeller is located in the chamber of the first container.At 2706, the entrance to the first container is closed or sealed with amicromotor. At 2708, the micromotor drives the impeller to circulate thematerial to be lysed and the lysing particulate material. The micromotormay drive the impeller for a sufficient length of time at a sufficientspeed until a desired or defined level of lysing has occurred.

FIG. 28 shows a method 2800 of operating a lysing apparatus according toone illustrated embodiment. The method 2800 may be particularly usefulfor use with the embodiment of FIG. 18.

At 2802, the micromotor may be removed from the entrance of the firstcontainer. At 2804, the material that has been lysed is removed from thechamber of the first container via the entrance.

FIG. 29 shows a method 2900 of removing material that has been lysedaccording to one illustrated embodiment.

At 2902, the material that has been lysed may be withdrawn using apipette.

FIG. 30 shows a method 3000 of operating a lysing apparatus according toanother illustrated embodiment.

At 3002, the micromotor may be reused with one or more additionalcontainers. It is noted that the micromotor, particularly when operatedat high speed, may not be particularly well protected from the materialto be lysed, lysing particulate material, or lysed material.Consequently, the micromotor may wear out. In many applications themicromotor may be employed to lyse multiple samples before failing.

FIG. 31 shows data on efficiency of lysis using an apparatus similar tothat of FIG. 4.

A first curve 3102 represents measured fluorescence versus time ofoscillation using an embodiment similar to that illustrated in FIG. 4.Fluorescence is proportional to the amount of nucleic acid released fromcells. A second curve 3105 represents measured fluorescence versus timeof oscillation using a commercially available “MINI-BEADBEATER-1 productfrom Biospec Products, Inc. of Bartlesville, Okla. As seen by comparisonof the first curve 3102 and second curve 3105, the embodiment of FIG. 4causes the release of cell contents more efficiently than thecommercially available apparatus.

FIG. 32 illustrates a dependency of lysis efficiency on the frequency.

A curve 3202 appears to indicate a nearly quadratic dependence of thedegree of lysis on frequency as controlled by changes to the appliedvoltage for a fixed amount of time.

FIG. 33 shows data representing spore lysis as a function of time for anembodiment similar to that illustrated in FIGS. 16 and 17.

The curves 3302, 3304 illustrate that the time to saturation iscomparable to that of the embodiments of FIG. 4, but with peakefficiency of only 80%. The power required for this efficiency was only400 mW, which is lower than the power used for various otherembodiments.

FIG. 34A shows a bidirectional flow system 3400 for lysing, capturing,and eluting a biological material according to one illustratedembodiment. As described in more detail herein, the bidirectional flowsystem 3400 may be operated to lyse a biological specimen, to capture abiological material on a particulate material therein, and to elute thebiological material therefrom. The system 3400 in FIG. 34A isparticularly suited to lysing biological specimens and controllingdirection and rate of flow, chemical compositions and physicalcharacteristics of fluids within a lysing chamber to induce capture ofbiological materials by and elution of such materials from a particulatelysing material therein. Efficient use of system 3400 for lysis, captureand elution surprisingly may simply require control of flow direction,pH and salt concentration of fluids within the system. Under certainconditions, controlling temperature of certain of the fluids may also beadvantageous.

In certain embodiments of the design of apparatus or systems herein, thedesign may include multiple modules, as further discussed below. Eachmodule may be useful for carrying out particular aspects of the methodsemploying such apparatus or systems. Modules may be independentlyfunctional, but in a system may be joined, for example by snappingtogether or via Luer-Lock connectors.

In certain embodiments, the design of an integrated modular system forlysis of specimens and capture and elution of biological material ofinterest therefrom is such that multiple functions are carried out bythe system. A system of such a design is particularly suitable forlysis, capture and elution of a biological material by control of fluidflow direction and chemical composition of the fluids within the systemduring different functions of the system or phases of operation.Functions of a system, such as that shown in FIG. 34A, exemplified bythe specimen being cells and the biological material being DNA, mayinclude the following: sample introduction; fluid movement; celldisruption and DNA capture; DNA elution; optionally heating and cooling;and optionally testing the isolated DNA. At least flow direction andchemical composition are controlled during each function.

In FIG. 34A, the bidirectional flow system 3400 for lysing, capturingand eluting a biological material includes a sample and elution module3434, a lysing module 3410, a syringe 3413, and optionally a heater3416. The sample and elution module 3434 includes a sample reservoir3402 and an elution buffer reservoir 3420. The lysing module 3410includes a lysing chamber 3412 and a micromotor 3430 coupled to animpeller 3431. During operation of the system 3400, the lysing chamber3412 contains fluid and particulate lysing material. The particulatelysing material may be densely packed within the chamber. The syringe3413 includes a barrel 3414 and a plunger 3415. The syringe 3413 may beoperated manually or driven automatically, for example by positioningthe syringe 3413 within a syringe pump (not shown in FIG. 34A). Syringepumps are commercially available, for example from New Era Pump Systems,Inc. (Wantagh, N.Y., USA) The heater 3416 may be positioned to heatfluid within the barrel 3414 of the syringe 3413.

The sample reservoir 3402 of the sample and elution module 3434 isfluidly communicatively coupled via conduit 3404, valve 3406 and conduit3408 to the lysing chamber 3412 of the lysing module 3410. In certainembodiments, the sample reservoir 3402 contains a biological specimensuspended or dissolved in a fluid having a chemical composition suitableto induce capture of a biological material by a particulate material inthe lysing chamber 3412 upon release of the biological material from thebiological specimen by lysis.

The elution buffer reservoir 3420 of the sample and elution module 3434is fluidly communicatively coupled via conduit 3418, valve 3406 andconduit 3408 to the lysing chamber 3412 of the lysing module 3410. Incertain embodiments, the elution buffer reservoir 3420 contains a fluidhaving a chemical composition suitable to induce release of biologicalmaterial from particulate material on which it has been captured in thelysing chamber 3412.

The micromotor 3430 is electrically communicatively coupled tocontroller 3432. The controller may control the speed, duration and/ortiming of operation of the micromotor, and thus the impeller 3431 withinthe lysing chamber 3412.

The lysing module 3410 is fluidly communicatively coupled to the barrel3414 of the syringe 3413. The plunger 3415 of the syringe 3413 ispositioned within the barrel 3414 of the syringe 3413. The plunger 3415of syringe 3413 may be manually or controllably slidably operable todraw fluid into the barrel 3414 by moving the plunger 3415 in adirection so as to increase the volume within the barrel 3414.Alternatively, the plunger 3415 of the syringe 3413 may be manually orcontrollably slidably operable to force fluid out from the barrel 3414by moving the plunger 3415 in a direction so as to decrease the volumewithin the barrel 3414. The barrel 3414 of the syringe 3413 isoptionally in thermal contact with the heater 3416. The heater 3416 may,for example, include a band of an electrically resistive material, forinstance a foil band. The heater 3416 may provide heat to the barrel3414 of the syringe 3413 by closing a switch 3426 to supply voltage froma source 3428 to the heater 3416. The level of heat supplied to thebarrel 3414 of the syringe 3413 may be controlled, for example via apotentiometer or some other controller (not shown in FIG. 34A).

The sample and elution module 3434 further includes an outlet tube 3422,fluidly communicatively coupled to the lysing chamber 3412 via conduit3408 and valve 3406. The outlet tube 3422 is provided as a conduit todispense eluted biological material into receptacle 3424 for furtheruse, such as for testing or analysis. The plunger 3415 of the syringe3413 may be operated manually or via a controller (not shown in FIG.34A), for example controlling the operation of a pump, to draw fluidtoward the barrel 3414 of the syringe 3413 at a first rate, to pushfluid away from the barrel 3414 of the syringe 3413 at a second rate,and/or to stop flow for a period of time. The first rate and the secondrate may be the same or different.

In a first phase of operation of system 3400, the plunger 3415 of thesyringe 3413 may be operated to draw biological specimen from the samplereservoir 3402 via the valve 3406 into and/or through the lysing chamber3412 into the barrel 3414 of the syringe 3413. In one embodimentthereof, the impeller 3431 may be operated while sample is drawn throughthe lysing chamber. In another embodiment, the impeller 3431 and/or theflow may be stopped intermittently. Further, the rate of flow or therate at which the impeller 3431 is operated may be varied. Control ofone or more of these variables may allow optimization of lysis of thebiological specimen and/or collection of the biological material fromthe biological specimen.

A chemical composition of a fluid containing a biological specimen inthe sample reservoir 3402 is selected to induce or permit capture ofbiological material from the biological specimen upon lysis of thebiological specimen in the lysing chamber 3412. For example, in certainembodiments for isolation of nucleic acids, particularly DNA, frombiological specimens, fluids in which the biological specimens may besuspended or dissolved within the sample reservoir 3402 in preparationfor lysis and binding of the nucleic acid by particulate material in thelysing chamber 3412 may have high salt concentrations and/or low pH.

In embodiments of specimen-containing fluids with high saltconcentrations, salt concentrations of such fluids in the samplereservoir 3402, suitable to induce binding of nucleic acid, particularlyDNA, to the particulate material in the lysing chamber 3412, may varyfrom about 200 μM to about 500 μM. In other such embodiments, saltconcentration of specimen-containing fluids suitable to induce bindingof a nucleic acid, particularly DNA, may range up to about 1000 μM. Inyet other such embodiments, salt concentration suitable to inducebinding of nucleic acids, particularly DNA, may range up to about 2000μM or greater.

In embodiments of specimen-containing fluids with low pH, pH of suchfluids in the sample reservoir 3402, suitable to induce binding ofnucleic acid, particularly DNA, to the particulate material in thelysing chamber 3412 may vary from about pH 3.5 to about pH 5. In othersuch embodiments, pH of the specimen-containing fluids to induce bindingof a nucleic acid, particularly DNA, may vary from about pH 3.75 toabout pH 4.25. In yet other such embodiments, pH of specimen-containingfluids to induce binding of nucleic acids, particularly DNA, may beabout pH 4.

For example, the beads can be coated with antibodies specific to markersfor pathogens, such as the enterotoxin B protein from Staphylococcusaureus. Similarly the beads can be coated with antibodies that bindmarkers for cancers, such as the p53 protein.

Also for example, in a “sandwich” approach, the beads can be pre-coatedwith a more universal receptor such as streptavidin therefore able tocapture reagents that are conferred with the corresponding ligand suchas biotin. Biotinylated antibodies are both readily available and easyto make. This approach allows one version of bead, such as streptavidincoated beads to serve as a generic product that can then be used tocapture any specific protein by linking with the correspondingbiotinylated antibody. The binding of the biotinylated antibody can becaptured on the bead either as a manufacturing process prior to sampleprocessing or by the user prior to processing a sample, or can beperformed as part of the sample processing, combining beads, antibodyand sample at the same time.

As a further example, an alternative “sandwich” approach to achieving a“universal” bead to capture specific protein is to pre-coat the beadwith an antibody specific to antibodies of another species, such asmouse anti-goat IgG, then use goat antibody specific to the protein ofinterest.

In a second phase of operation of system 3400, the plunger 3415 of thesyringe 3413 may be operated to push fluid out of the barrel 3414 of thesyringe 3413 back through the lysing chamber to return specimen to thesample reservoir 3402 after capture or binding of the biologicalmaterial therefrom. Nonspecific capture of DNA on the beads may occurduring both the forward and the reverse passage of the sample throughthe lysing chamber. For sequence specific capture, the fluid in thebarrel 3414 of the syringe 3413 may be heated and cooled beforereversing the direction of flow, as further discussed below. The fluidin the sample reservoir at the end of the capture process is wastematerial or material that contains analytes of a different chemistryother than what is captured by the beads. So for example DNA may becaptured on the beads and proteins that have not been exposed todenaturant can be returned to the sample chamber to then be analyzed fora specific protein. A quantity of air may be initially drawn into thebarrel 3414 of the syringe 3413 at the beginning of the first phase ofoperation and used to force all of the waste fluid from the lysingchamber and the conduits into the sample/waste reservoir at the end ofthe first phase of operation.

In certain embodiments, the system may include particulate materialhaving antibodies attached thereto to capture analyte protein orpolypeptide markers while other particulate material remains dedicatedto capturing nucleic acids. Further, labeled antibodies may beintroduced during lysis and capture, for example, to label the proteinor polypeptide. After capture, the labeled antibody may be eluted, forexample with Urea. In a certain embodiment, for example, if a portion ofthe beads are designed for DNA capture and another portion for proteinor polypeptide capture, capture of both may occur at the same time. Insuch an embodiment, the DNA may be elute with low salt and the proteinor polypeptide with urea.

Beads that are design for captured of proteins can be manufacturedseparately from beads that are designed for capture of nucleic acid.After the separate manufacturing processes are complete then the twobead types can be combined in useful proportions into one cartridge thatis then able to process proteins and nucleic acids. The beads that aredesigned for protein capture can be conferred with protein specificantibodies, or with protein specific aptamers, or with a universalreceptor such as streptavidin, or with a binding agent that bindsproteins that have been “engineered” for specific capture. This caninclude using Nickel coated bead used to capture proteins with histidinetails.

Beads that are design for capture of nucleic acid can be generic,binding essentially all nucleic acids such as the silica or zirconiabeads. Alternatively these beads can be conferred with specific nucleicacids (capture probes) that bind specific analytes by hybridization.Specific nucleic acid capture can also be facilitated by the sandwichapproach by conferring the beads with a “universal” capture probe, andthen providing a linker probe that has two domains, one domaincomplimentary to the analyte and the other domain complimentary to the“universal” capture probe.

Many combinations of beads for nucleic acids, specific or generic and becombined with beads for proteins, specific or generic with a singlecartridge.

In a third phase of operation of system 3400, elution buffer may bedrawn into the barrel 3414 of the syringe 3413 from the elution bufferreservoir 3420 via valve 3406 through lysing chamber 3412. As above, theflow may be continuous or intermittent and the rate and/or volume may bevaried to optimize elution of biological material from the particulatelysing material. Further, the barrel 3414 of the syringe 3413 may beheated at this stage as well, for example to heat the elution fluid tooptimize elution of captured biological material from the particulatematerial in the lysing chamber.

In certain embodiments of systems and methods for isolation of nucleicacids, particularly DNA, buffers for elution of the nucleic acids fromthe particulate material in the lysing chamber may have a lower saltconcentration and/or a higher pH than the salt concentration and pH ofthe fluid in which the biological specimen containing the nucleic acidswas suspended or dissolved to induce binding of the nucleic acids,particularly DNA, to the particulate material, as described above.

In embodiments of sample elution buffers with lower salt concentrations,the salt concentrations of the elution buffer placed in and drawn fromthe elution buffer reservoir 3420 may be less than about 200 μM. Inother such embodiments, the salt concentrations of the elution buffersmay be less than about 200 μM. In yet other such embodiments, the saltconcentrations of the elution buffers may be less than about 10 μM. Inparticular such embodiments, the elution buffers may contain no salt.

In certain embodiments of sample elution buffers with higher pH, the pHof the elution buffers may be greater than about pH 5. In other suchembodiments, the pH of the elution buffers may be greater than about pH6. In yet other such embodiments, the pH of the elution buffer may begreater than about pH 7. In further such embodiments, the pH of theelution buffer may be greater than about pH 8.

In a fourth phase of operation, the eluted material may be pushed fromthe barrel 3414 of the syringe 3413 and the lysing chamber 3412 viaconduit 3408, valve 3406 and outlet tube 3422 into receptacle 3424 forfurther manipulation or use, including analysis or testing. Air mayagain be expelled from the barrel 3414 of the syringe 3413 to force allof the fluid containing the biological material from the system and intothe receptacle. Instead of collecting the eluate in a receptacle, theoutlet tube may alternatively be connected directly to a testingapparatus in which the eluted material may be analyzed immediately uponelution. Outlet tubes may have narrow capillary sized channels to allowcareful metering of the dispensed fluid, if necessary. The outlet mayalso include fluid sensing systems to monitor fluid flow and to providefeedback to the control system to control operation of the syringe 3413.

Use of a syringe 3413 to provide flow within the described lysis,capture and elution system provides an efficient contamination freeapproach, since the syringe 3413 can be readily and inexpensivelyreplaced after each use of the system. While syringe pumps have beendisclosed for use in the systems and methods disclosed herein, othertypes of pumps may be used, for example peristaltic pumps or other typesof metering pumps. A particular advantage to use of syringe pumps orperistaltic pumps is that the syringe or tubing for the peristaltic pumpis disposable and may be discarded along with the lysis chamber aftereach use. This provides a simple configuration for efficientlyprocessing samples without concern for cross-contamination from onesample to the next. Although elimination of wash steps is a distinctadvantage of the systems and methods disclosed herein, one or more washsteps could be included, if desired in particular embodiments, with washfluid being provided via either the sample reservoir or the elutionbuffer reservoir.

Analysis or testing of biological materials obtained by systems andmethods described herein may include, for example, subjecting DNA toamplification by PCR. In certain such procedures, the heater 3416 may beadvantageously used. For example, in using the system 3434 to provideDNA for testing involving enzymatic amplification such as by PCR, theheater 3416 may supply heat to denature the DNA to form single strandsin the barrel 3414 of the syringe 3413 prior to dispensing into thereceptacle 3424. The heat-denatured DNA may then be cooled while stillin the barrel 3414 of the syringe 3413 to prevent re-association of thestrands. Such heat-denatured DNA may be particularly useful in enzymaticamplification reactions such as by PCR.

FIG. 34B shows positions of valve 3406 during certain phases ofoperation of system 3400 in FIG. 34A according to one illustratedembodiment.

Valve position 3406 a in FIG. 34B may be employed for two phases ofoperation. One phase corresponds to withdrawing biological specimen fromsample reservoir 3402, via conduit 3404, valve 3406 and conduit 3408into lysing chamber 3412 and barrel 3414 of syringe 3413. The otherphase of operation using valve position 3406 a corresponds to pushingspecimen remaining after lysis and collection from barrel 3414 ofsyringe 3413 and lysing chamber 3412 back into sample reservoir 3402 viaconduit 3408, valve 3406 and conduit 3404.

Valve position 3406 b in FIG. 34B may be employed for a further phase ofoperation. This phase, an elution phase, corresponds to withdrawingelution buffer from elution reservoir 3420 via conduit 3418, valve 3406and conduit 3408 into lysing chamber 3412 and barrel 3414 of syringe3413.

Valve position 3406 c in FIG. 34B may be employed for yet another phaseof operation. This phase, a dispensing phase, corresponds to pushingeluted biological material from barrel 3414 of syringe 3413 and lysingchamber 3412 into receptacle 3424 via conduit 3408, valve 3406 andoutlet tube 3422.

Cellular samples for use in the systems disclosed herein, such as inFIG. 34A, may be obtained from a source such as a human or animal by useof swabs. Swabs may also be used to collect samples for testing fromvarious surfaces that may hold cellular samples of interest. Thecellular material collected on the swabs may then be suspended in fluid.Appropriate buffer, salts and mild detergent may be present in the fluidin order to drive the capture of DNA by the beads in the lysing chamberduring lysis of the cells. The fluid sample containing the cells isplaced, for example, in the sample reservoir in the sample and elutionmodule of the system shown in FIG. 34A.

For example, binding of nucleic acid can be facilitated by cationsprovided by either sufficiently low pH, as low as 3.0 or sufficientlyhigh enough concentration of salt such as NaCl, from 200 mM to 5 M, orLithium Chloride, or ammonium Sulfate, or ammonium acetate. Chaotropicsalts can be used up to 6 M.

High salt concentrations facilitate both non-specific binding of DNA tosilica surfaces and sequence specific capture (hybridization) of DNA.

Detergents in the binding buffer may be used to reduce non-specificbinding, such as Sodium Dodecyl Sulfate (SDS). If the detergent iscompatible with nucleic amplification reactions such as PCR, then theneed to wash away the detergent before elution of DNA can be avoided.These detergents are typically non-ionic and can include Tween, Triton,and Nonidet. If the salt that is used to facilitate binding the DNA isorganic it may also serve to reduce non-specific binding, such asacetate as the anion or guanidinium as the cation.

Solutions that facilitate release and elution of DNA are low in ionicstrength such as water, are typically buffered by concentration ofbuffer such as Tris at 10 mM to 30 mM. If the binding buffer has a lowpH, such as pH 3 to 5 then it can help to neutralize it with a raised pHin the elution buffer, to include a range of pH 8 to 10. This will serveto facilitate release as well as compatibility with subsequent reactionsof nucleic acid amplification.

Nucleic acids, in particular DNA, may be bound nonspecifically to theparticulate lysing materials, as noted elsewhere herein. Alternatively,certain aspects of the materials and methods described herein may beadapted for sequence specific capture of nucleic acids. Sequencespecific capture methods may be advantageous when nonspecific captureresults in the capture of biological materials in addition to those ofinterest. For example, soil or stool samples may contain anionicpolysaccharides that may be nonspecifically captured in addition tonucleic acids when using the systems and methods disclosed herein. Inaddition, stool samples may contain a variety of PCR inhibitors that maybe nonspecifically bound and may then co-purify with nucleic acids,e.g., DNA. An alternative to using particulate materials thatnonspecifically bind DNA and such other materials is to incorporatesequence specific capture probes on the particulate lysing materials.These probes would be selected on the basis of specificity for bindingcertain sequences in the nucleic acid(s) of interest. Such use ofsequence specific capture would not only eliminate binding ofnon-nucleic acid materials, but would also eliminate binding of hostnucleic acids from host cells gathered on the swabs. Sequence specificcapture methods may thus increase sensitivity. The particulate materialsor beads may be functionalized by a variety of methods for use insequence specific capture.

For example, beads may be conferred with capture probes that bind thepolymerase gene of HIV. The target specific sequence may range in lengththat facilitate sufficient affinity and specificity for the analyte,such as but not limited to 10 to 60 nucleotides long. The capture probescan further be designed with degeneracies, to facilitate capturing allor most variations of the analyte.

When using sequence specific capture, certain aspects of the methods forlysis, capture and elution, as described above, may vary. For example,in order to expose sequences in the DNA for specific binding by theprobe, the double-stranded genomic DNA must be denatured to yieldsingle-stranded DNA. This can be done by heat denaturation. Thus, whenthe specimen is passed through the lysing chamber and into the syringe,the syringe may then be heated to denature the DNA that has not alreadybound to the beads in the lysing chamber. The heating denatures the DNAto single-stranded DNA, which may then be cooled to inhibitre-annealing. In this manner the sequences of the heat denaturedsingle-stranded DNA are exposed to allow specific binding toprobe-containing beads during passage of the heated/cooled specimen backthrough the lysing chamber.

In certain embodiments, a system for lysing biological specimens andcapturing and eluting biological material may be a lysing module havinga lysing chamber containing particulate lysing material, e.g., beads, amicromotor-driven impeller and two syringes—a first syringe and a secondsyringe—each having a barrel fluidly communicatively coupled to thelysing chamber of the lysing module. The barrel of either one or both ofthe syringes may further have a heater in thermal contact therewith.Such a system may operate similarly to the system described above. Forexample, biological specimen may be pumped from a barrel of the firstsyringe into and through the lysing chamber into a barrel of the secondsyringe. The waste sample may then be pumped from the barrel of thesecond syringe back through the lysing chamber into the barrel of thefirst syringe or, if the first syringe has been removed, directly into awaste receptacle. A third syringe having a barrel containing elutionbuffer may then be substituted in place of the first syringe. Theelution buffer may be delivered from the barrel of the third syringethrough the lysing chamber into the barrel of the second syringe. Thethird syringe may then be either removed or replaced by a fourth syringehaving a barrel. The elution buffer may then be pumped from the barrelof the second syringe back through and out of the lysing chamber andeither into a receptacle or into the barrel of the fourth syringe. Thebarrel of the second syringe may be heated as described above, that is,during the lysis and capture phase and/or during the elution phase. Eachsyringe may simply be operated manually or may be operatedautomatically, for instance via a syringe pump, a motor and linkage, asolenoid, or other actuator.

During use of the systems described herein, the volume of sample passedthrough the lysing chamber for cell disruption and capture of biologicalmaterials, in particular DNA may be many times the volume of the lysingchamber. In certain embodiments, volumes of sample may range from 200 μlto 2000 μl. In certain particular embodiments of the systems and methodsdisclosed herein, the lysing module may be an OmniLyse™ (OL™) lysingapparatus. In particular embodiments using the OL™ lysing apparatus,processing samples of a volume noted above may take between about 15 andabout 120 seconds.

The embodiment of the system and methods disclosed herein shown in FIGS.34A and 34B and described in detail above exemplifies in a non-limitingmanner isolating biological materials from biological specimens lysedusing an impeller apparatus within the lysing chamber 3412. However, oneof skill in the relevant art will recognize that the system exemplifiedin FIGS. 34A and 34B and its use could readily be adapted to isolationof biological materials that are released from biological specimenslysed by other means. For example, as described in detail elsewhereherein, such biological specimens may be lysed by oscillation of alysing chamber. Further, lysing of specimens could be carried outultrasonically, or could even include some other type of physical orchemical treatment, so far as the method of treatment yields abiological material having a chemical composition that is appropriatefor binding of the biological material to particulate material withinthe lysing chamber and so far as the biological material captured by andeluted from the particulate material is suitable for further use oranalysis.

FIGS. 35A and 35B show a lysing apparatus 3500, according to anotherillustrated embodiment.

The lysing apparatus 3500 includes a body 3502 that forms a chamber3504. The body 3502 may have an opening 3506 sized and dimensioned toreceive an impeller 3508 therethrough such that the impeller resides inthe chamber 3504. The opening 3506 may optionally receive part or all ofa drive motor, for instance a micro electric motor 3510. The electricmotor 3510 is coupled to drive the impeller 3508. The electric motor3510 is selectively operable in response to power supplied thereto. Theelectric motor 3510 may be secured in the opening 3506 via a press typefitting or interference fit. In particular, an inner wall forming theopening 3506 and/or chamber 3504 may be slightly tapered to sealingengage a side wall of the electric motor 3510 as the electric motor isadvanced through the opening 3506 and into the chamber 3504.Alternatively, or additionally, a side wall of the electric motor 3510may be slightly tapered to sealing engage a side wall of the opening3506 and/or the chamber 3504 as the electric motor 3510 is advancedthrough the opening 3506 and into the chamber 3504. Alternatively, theelectric motor 3510 and the opening 3506 and/or chamber 3504 may includecoupler structures. For instance, the electric motor 3510 and theopening 3506 and/or chamber 3504 may include threads (not shown) whichsealing mate together as the electric motor 3510 is advanced through theopening 3506 and into the chamber 3504. Alternatively, a bayonet (notshown) or lug type (not shown) coupler structure may be employed. Othersealing structures may be employed. For example, one or more gaskets,washers or O-rings (not shown) may be employed, with or without a seator peripheral ring to seat the gasket, washers or O-rings. The seal maybe a fluid tight seal and/or a gas tight seal.

The lysing apparatus includes a first port 3512 a and a second port 3512b (collectively 3512). The first and second ports 3512 include passages3514 a, 3514 b, respectively, (collectively 3514) to provide fluidcommunication with the chamber from an exterior thereof. The ports 3512may be used to as input ports to supply material to the chamber 3504and/or as output ports to remove material from the chamber 3504.

Each port 3512 may have a coupler 3516 a, 3516 b (collectively 3516)that allows selective coupling to the respective port 3512 a, 3512 b.For example, each of the ports 3512 may include a respective Luer-Lock®fitting or Luer-Slip® fitting, male or female. The Luer-Lock® orLuer-Taper® fittings allow the coupling of syringes 3518 a, 3518 b (FIG.35A, collectively 3518) to the lysing apparatus 3500. For example, afirst syringe 3518 a may be coupled to the first port 3512 a to allowsample or specimen injection, while a second syringe 3518 b may becoupled to the second port 3512 b to allow removal of a sample orspecimen after lysing (i.e., lysed material). Such may allow the passageof a sample or specimen back and forth through the chamber 3504, forinstance to enhance performance of the lysing or of DNA capture. Use ofsyringes 3518 may occur at either port 3512 a, 3512 b or at both ports3512. The advantages of using a syringe 3518 as a sample or specimendelivery system include the fact that syringes 3518 are inexpensive,disposable, and employ positive displacement of fluid for a high degreeof reliability in rapidly dispensing volumes. The Luer-Lock® designexemplifies a universal attachment that seals reliably and mates withmany devices that also have complimentary Luer-Lock® fittings.

As illustrated in FIG. 36, selectively fastenable fittings, such as theLuer-Lock® fittings, may allow multiple lysing apparatus 3500 a-3500 b(collectively, 3500, only three illustrated) to be connected insuccession. Such may advantageously be used to sequentially process asample or specimen through multiple stages. Additionally, oralternatively, lysing particulate (e.g., beads) in the differentsequential lysing apparatus 3500 may each have a respective receptivityfor different molecules. For instance, the particulate in successiveones of the sequential lysing apparatus may be conferred with receptors(e.g., binding sites) to capture different respective molecules from thesame sample or specimen. Each lysing apparatus 3500 with a differentcaptured molecule, may then be easily separated from one another, andprocessed individually using different types of elution acts or steps.

FIGS. 37A and 37B show a lysing manifold or array 3700, according to oneillustrated embodiment. The lysing manifold or array 3700 includes ablock or frame 3702 that has a plurality of positions 3704 a, 3704 h(collectively 3704, only two called out in FIG. 37A) to hold respectiveones of one or more individual lysing apparatus 3706 a-3706 h(collectively 3706, six illustrated). The individual lysing apparatus3706 may, for example, take the form of distinct lysing apparatus whichemploy a chamber that receives an impeller and electric motor, forinstance, the individual lysing apparatus 37006 may be identical orsimilar to the lysing apparatus 3500 (FIG. 35). Each individual lysingapparatus 3706 may include a respective disposable electric motorcoupled to drive the impeller. Each individual lysing apparatus 3706 mayinclude a first port 3708 a and a second port 3708 b (collectively 3708,only two called out in FIG. 37A). The ports 3708 may function as inletand/or outlets to a chamber (not called out in FIG. 37A or 37B).

As illustrated in FIG. 37B, the lysing manifold or array 3700 mayinclude a support structure 3710 to support one or more blocks or frames3702 and associated individual lysing apparatus 3706. In particular, thesupport structure 3710 may include rails 3710 a, 3710 b to hold theblock or frame 3702 and associated individual lysing apparatus 3706positioned relative to a structure that receives the lysed material, forexample a plate such as a micro-titer plate 3712. For instance, thesupport structure 3710 may hold the block or frame 3702 such that theassociated individual lysing apparatus 3706 are positioned aboverespective ones of a plurality of wells 3712 a (only one called out inFIG. 37B) of the micro-titer plate 3712. FIG. 37B shows only a singlelysing manifold or array 3700 carrying a single row of individual lysingapparatus 3706, constituting a one-dimensional array of lying apparatus3706. Alternatively, the support structure 3710 may carry additionallysing manifolds or arrays, each carrying a respective single row ofindividual lysing apparatus 3706. The individual lysing apparatus 3706carried by the plurality of lysing manifolds or arrays 3700 canconstitute a two-dimension array. As a further alternative, a singlelysing manifold or array 3700 may carry individual lysing apparatus 3706arranged in a two-dimensional array. As an even further alternatively, amotor and drive mechanism may be coupled to move a single lysingmanifold or array 3700 carrying the individual lysing apparatus 3706along the rails 3710 a, 3710 b of the support structure 3710. Thus, theone-dimensional array of lysing apparatus 3706 may be moved to address atwo-dimensional array of positions. Movement may be controlled manuallyor automatically, for example via one or more computer processors,motors, actuators and or transmissions.

As described immediately above, individual lysing apparatus 3706 can bebundled together into a lysing manifold or array 3700 (e.g., one- or twodimensions) to facilitate multiplex processing. The distance betweencenters for these individual lysing apparatus 3706 can, for example, be9 mm or a multiple of 9 mm to match a standard format of a micro-titerplate 3712 (e.g., with 9 mm spacing, 96 well plate or greater).Similarly, the use of electric motors with diameters below 4.5 mm allowsthe manifold or array of lysing apparatus 3700 to be used formicro-titer plate formats with 4.5 mm spacing (e.g., 384 well plate).Bundling the individual lysing apparatus 3706 in strips or rows of 4, 8,6 or 12 may facilitate use for automated or semi-automated processing ofsamples in a micro-titer format. Additionally, if intake ports 3708 a ofthe individual lysing apparatus 3706 are designed to receive sample orspecimen from pipette tips, then the individual lysing apparatus 3706may be addressed by multichannel pipettors for either manual or roboticoperation. The block or frame 3702 may be fabricated monolithically froma single block of material that has been molded or cut-extruded withmultiple sites for the individual lysing apparatus 3706.

The flow through nature of some embodiments may allow for reuse of thesystem for processing additional samples or specimens. For example, theflow through nature may facilitate performance of one or more wash actsor steps to sterilize or otherwise sanitize or cleanse the system.Containers may be reused by cleaning and/or sterilizing the containerbetween uses. This may be coordinated with downstream processing of onesample or specimen such that the container may be made ready for anothersample or specimen during the downstream processing. One or more actsmay be employed to clean and/or sterilize the container, for exampleusing a high pH or low pH solution, bleach, detergent or combinationsthereof. Adjusting pH may advantageously reduce the number of wash actsor steps, since the pH can be easily neutralized. An alternativeapproach may be the use of di-ethyl-pyrocarbonate (DEPC). DEPC compoundcan destroy proteins and nucleic acid. This treatment may be followed bya single wash and then a flow of hot air. Because DEPC is so volatile,it may be removed by degradation and evaporation during the act ofpassing heated air over any surfaces treated with the DEPC.

The various embodiments, whether flow through or not, may allow foranalyte capture through various mechanisms either within the samechamber in which lysis occurs and/or in other chambers, for instancechambers arranged subsequently with respect to a flow of sample orspecimen. As explained herein the flow through systems or apparatus(e.g., oscillating arcuate motion based or rotational impeller based)can be combined with analyte capture by using the same particulatematter (i.e., lysing particulate matter) used to perform lysis tocapture analyte molecules within the same chamber. Additionally, oralternatively, lysis can also be combined with an act or step that usesanother mechanism for analyte capture that may normally follow the lysisact. This approach still advantageously obviates the use of harshreagents, as well as eliminating the associated need to perform washacts or steps prior to any subsequent enzymatic reaction. For example, 1μm magnetic particles can be combined with the sample or specimen beforeor after disruption (i.e., lysing). Thus, DNA capture can occur on thesemagnetic particles after the disruption has been accomplished by largerlysing particulate or beads. This principle may also be applied to othernon-chemical approaches to cell lysis, such as sonication, where capturemay occur on an additional surface at the same time or followingsonication. Such may still allow wash acts or steps to be avoided beforeany enzymatic reaction.

Flow through configuration and high energy mixing of lysing particulateor beads may provide distinct advantages in binding and elutionkinetics. By passing the sample or specimen through a relatively smallchamber with a dense suspension of lysing particulate or beads that areactively shaken up or stirred, induces a high rate of “collisions”between analyte DNA and the lysing particulate or beads. This may besimilar is some respects to passing a sample or specimen through anaffinity filter, providing a stark advantage over the common approach ofusing micron-sized magnetic beads. Such magnetic beads typically must bedispersed throughout the entire sample or specimen volume.

The interface between a solid phase and a solution phase has a boundarylayer in the liquid phase that is relatively static and does notparticipate in active mixing. The molecules in the solution phase mustdiffuse through this layer to reach the surface of the solid phase.Diffusion is a slow process compared to turbulent mixing and convection.As the energy of motion of the lysing particulate or beads increases,the extent of active mixing increases, reducing this boundary layer andserving to increase the rate of arrival of DNA toward the bead surface,thus accelerating the binding. Alternatively, when eluting the DNA, thehigh energy motion of the lysing particulate or beads can alsoaccelerate the dissociation of the DNA from the lysing particulate orbeads by serving to escort the DNA away from the lysing particulate orbeads and the boundary layer. This can be especially helpful in thecommon case where the DNA binding system has a large binding capacity tocapture a large load DNA, e.g., 5 to 60 μg, but is also useful incapturing very low loads of analyte DNA. The high binding capacity willnormally reduce the efficiency of elution for low copy numbers byoffering so much surface area to re-bind with and so much boundary layerthat can retain some of the DNA. The shear forces generated by the highenergy motion generated by the oscillating arcuate motion basedembodiments (FIGS. 1A-5) or the rotational impeller based embodiments(FIGS. 16-18) can contribute to dissociating DNA from the surface and toescorting DNA from the reduced boundary layer, enabling the DNA to jointhe bulk fluid that is dispensed out of the chamber during the elutionact or step.

At least some of the embodiments described herein may enable a“sandwich” type of detection scheme using the lysing particulate orbeads. This principle can apply to proteins and to nucleic acids, aswell as other ligand/receptor combinations. In the case of nucleicacids, a DNA capture scheme such as a branched DNA assay developed atChiron can be performed on the lysing particulate (e.g., beads) used tolyse cells and bind DNA in either the oscillating arcuate motion basedapparatus (i.e., bead beater) or the rotational impeller based apparatus(i.e., bead blender). Analyte nucleic acid can be captured byheterobifunctional extender probes that can crosslink analyte sequenceto capture probes that are attached to the lysing particulate or beads.Subsequent layers of branched DNA assay can be added to the capturedanalytes on the lysing particulate or beads. Each layer of probe can beaccompanied with an adjusted speed of lysing particulate or bead mixingto mediate between the acceleration of binding of each layer and the DNAscission that is associated with the high shear forces. Building thebranched DNA scheme on these lysing particulate or beads can facilitaterapid binding kinetics for each layer of binding including the captureof analyte. This can also facilitate very efficient concentration ofanalyte from very large volumes and can facilitate efficient wash actsor steps between binding acts or steps.

After an entire complex of probes of the branched DNA scheme has beenbuilt on the lysing particulate or beads, there are at least three waysto proceed to detection. One is to detect within the container orcartridge of the lysing apparatus (e.g., bead blender). In the case ofchemiluminescent detection, mixing can occur during the detection act orstep. Another approach is to dispense the lysing particulate or beadsinto a well or tube for detection. A further approach is to release theDNA complex from the lysing particulate or beads and elute the reportergroups into a well or tube for detection. This approach has thepotential advantage of providing a better ratio of analyte specificsignal to background signal by enabling a release act or step that is infact analyte specific. For example, one or more layer of probes may bereleased by cleaving with light at photo-cleavable junctions. Eluting inhigh pH, or in low salt with heat can denature the DNA and therebyrelease the reporter group. Nucleases may be used to release the DNAcomplex at specific sites or non-specifically. The rapid movementprovided by the oscillating rotational motion based apparatus (i.e.,bead beater) or the rotational impeller based apparatus (i.e., beadblender) may enhance the efficiency of the release or elution act orstep.

As will be recognized by those of ordinary skill in the art based on theteachings herein, the use of lysing particulate or beads to lyse cellsand to extract DNA can be performed using any surface chemistry that hasa nonspecific affinity for DNA. This can include using silica or silicalike beads and employing high salt or low pH to induce DNA to bind tobeads followed by application of a low salt solution to elute the DNAaccording to the Boom method. The can also include using negativelycharged beads that when accompanied by a solution of divalent cationssuch as Mg++, will form a salt bridge between the beads and DNA. Thisapproach also uses low salt and EDTA to elute the DNA. Such is describedat Hawkins, et al, Nucleic Acids Res. 1995, 23:22. This may also includecapturing and releasing DNA with anion exchange resins on silica lysingparticulate or beads. A resin such as diethylaminoethanol that containsa tertiary amine can bind DNA in low pH and can then elute DNA in thepresence of a medium to high salt concentration.

FIGS. 38A and 38B show a lysis apparatus in the form of a stopcock valve3800, according to yet another illustrated embodiment. The stopcockvalve 3800 includes an outer body portion 3802 that forms a receptacleor outer chamber 3804. The stopcock valve 3800 includes an inner bodyportion 3806 that forms a receptacle or inner chamber 3808. The stopcockvalve 3800 also includes an impeller 3810 received in the inner chamber3808 for rotation therein. The stopcock valve 3800 further includes anelectric motor 3812 coupled to drive the impeller. The stopcock valve3800 may additionally include a handle 3818 or other engageablestructure to allow a torque to be applied to the inner body portion 3806to allow such to be easily rotated or pivoted with respect to the outerbody portion 3802.

The inner body portion 3806 is rotatable (double headed arrow 3807) inthe inner chamber 3808 about a longitudinal axis 3809 to open and closea plurality of ports 3820 a-3820 c (collectively 3820), therebyproviding or shutting off fluid communication with the inner chamber3808. Such may be employed to allow the passage of different fluidsthrough the inner chamber 3808 at different times during the operationof the stopcock valve lysis apparatus 3800 and/or for defining differentflow paths through the stopcock valve lysis apparatus 3800. Thestructure may ensure that one or more ports in the wall of the innerchamber 3808 fluidly communicate with a port, channel or passage throughthe outer body portion 3802. Thus, a port 3820 may be aligned with achannel to a pump, while at least one other port 3820 in the fluidlycommunicates with a channel containing a fluid intended to pass throughthe inner chamber 3808. This design enables the stopcock lysis apparatus3800 to integrate with other chambers that serve other functions orwhich hold other fluids, such as a mix of the sample and binding buffer,elution buffer, pump, a waste reservoir, and/or chamber foramplification and detection.

FIGS. 39A-39C show a lysis apparatus 3900, similar to that of FIGS. 38Aand 38B. In particular, FIG. 39A is an exploded view of the lysisapparatus 3900, FIG. 39B is assembled view, and FIG. 39C shows a portionof the lysis apparatus 3900 rotated 180 degrees from an orientationillustrated in FIG. 39A.

The lysis apparatus 3900 may include an outer vessel 3902 that forms anouter chamber 3904, an inner vessel 3906 that forms an inner chamber3908, an impeller 3910 and an electric motor 3912 coupled to drive theimpeller 3910. The outer chamber 3904 is sized to receive the innervessel 3906 therein. For example, the outer chamber 3904 may have aninner peripheral or inner diameter D₁ that closely receives an outerperiphery or outer diameter D₂ of the inner vessel 3906. Such may besufficiently closely received as to form a fluid tight or a gas tightseal therebetween. Additionally, or alternatively, one or more gaskets,washers or O-rings may be employed. Additionally, or alternatively, acoupler structure (e.g., threads) may be employed. The outer chamber3904 is open at one end 3914 a, to receive the inner vessel 3906. Theouter chamber 3904 may be closed at the opposite end 3914 b. The innerchamber 3908 is open at one end 3916 a, to receive the impeller 3910and, optionally electric motor 3912. The inner chamber 3908 may be openat the opposite end 3916 b (as illustrated in FIGS. 39A and 39C), forexample where the outer chamber 3904 is closed at the opposite end 3914b. Alternatively, the inner chamber 3908 may be closed at the oppositeend 3916 b (as illustrated in FIGS. 40A and 40B).

The inner chamber 3908 is sized to receive the impeller 3910, andoptionally part or all of the electric motor 3912 therein. For example,the inner chamber 3908 may have an inner peripheral or inner diameter D₃that closely receives an outer periphery or outer diameter D₄ of theelectric motor 3912. Such may be sufficiently closely received as toform a fluid tight or a gas tight seal therebetween. Additionally, oralternatively, one or more gaskets, washers or O-rings may be employed.Additionally, or alternatively, a coupler structure (e.g., threads) maybe employed. The inner vessel 3902 may include one or more protrusionsor other engageable structure 3918 a, 3918 b (collectively 3918)extending outwardly therefrom. Such allows the inner vessel 3906 to beeasily rotated within the outer vessel 3902.

As best illustrated in FIG. 39A, the outer vessel 3902 has a number ofports 3920 a-3920 c (collectively 3920) to provide fluid communicationsor a fluid path between the outer chamber 3904 and an exterior of theouter vessel 3902. Three ports 3920 a-3920 c are shown in theillustrated embodiment, although fewer or greater number of ports may beemployed. Likewise, the inner vessel 3906 has a number of ports 3922a-3922 c (collectively 3922) to provide fluid communications or a fluidpath between the inner chamber 3908 and an exterior of the inner vessel3906. Three ports 3922 a-3922 c are shown in the illustrated embodiment,although fewer or greater number of ports may be employed. The ports3920, 3922 arranged such that selected ports on the inner vessel 3906align with selected ports on the outer vessel 3902 in a firstorientation or configuration (FIG. 39A), while selected ports on theinner vessel 3906 align with selected ports on the outer vessel 3902 ina second orientation or configuration (FIG. 39C), different from thefirst orientation or configuration. The fluid path can be modified bysimply orienting the inner vessel 3906 with respect to the outer vessel3902. For example, in the illustrated embodiment, a first fluid pathbetween ports 3920 a, 3920 b of the outer vessel 3902 is establishedwhen the inner vessel 3906 is in a first orientation (FIG. 39A) withrespect to the outer vessel 3902. Rotating the inner vessel 3906 withrespect to the outer vessel 3902, for instance 180 degrees about alongitudinal axis (orientation illustrated in FIG. 39C), establishes asecond fluid path between ports 3920 a, 3920 c of the outer vessel 3902.Notably, the inner wall or periphery that forms the outer chamber 3904selectively seals one of the ports 3922 a, 3922 c of the inner vessel3906 depending on the orientation of the inner vessel 3906 with respectto the outer vessel 3902. Other embodiments may employ additional ports.Ports may be oriented at angles other than 180 degrees from one another.Thus, for example, the inner vessel 3906 may have ports oriented at 90degrees, 60 degrees or 45 degrees from each other. Such may provide agreater number of selectively selectable fluid paths. While the outerand inner vessels 3902, 3906, respectively, are illustrated having anequal number of ports 3920, 3922, respectively, in some embodiments thenumber of ports of the outer and inner vessels 3902, 3906 may not beequal to one another.

FIGS. 40A and 40B show a show a lysis apparatus 4000, similar to that ofFIGS. 38A and 38B. In particular, FIG. 40A is an exploded view of thelysis apparatus 4000 and FIG. 40B shows a bottom of the lysis apparatus4000.

The lysis apparatus 4000 may include an outer vessel 4002 that forms anouter chamber 4004, an inner vessel 4006 that forms an inner chamber4008, an impeller 4010 and an electric motor 4012 coupled to drive theimpeller 4010. The outer chamber 4004 is sized to receive the innervessel 4006 therein. For example, the outer chamber 4004 may have aninner peripheral or inner diameter that closely receives an outerperiphery or outer diameter of the inner vessel 4006. Such may besufficiently closely received as to form a fluid tight or a gas tightseal therebetween. Additionally, or alternatively, one or more gaskets,washers or O-rings may be employed. Additionally, or alternatively, acoupler structure (e.g., threads) may be employed. The outer chamber4004 may be open at both ends 4014 a, 4014 b, to receive the innervessel 4006. The inner chamber 4008 is open at one end 4016 a to receivethe impeller 4010 and, optionally electric motor 4012. The inner chamber4008 is closed at the opposite end 4016 b which thus closes the oppositeend 4014 b of the outer chamber 4004. The inner vessel 4006 has one ormore protrusions or other engageable structure 4018 extending from theopposite end 4016 b thereof. Such allows the inner vessel 4006 to beeasily rotated within the outer vessel 4002.

The inner chamber 4008 is sized to receive the impeller 4010, andoptionally part or all of the electric motor 4012 therein. For example,the inner chamber 4008 may have an inner peripheral or inner diameterthat closely receives an outer periphery or outer diameter of theelectric motor 4012. Such may be sufficiently closely received as toform a fluid tight or a gas tight seal therebetween. Additionally, oralternatively, one or more gaskets, washers or O-rings may be employed.Additionally, or alternatively, a coupler structure (e.g., threads) maybe employed.

As best illustrated in FIG. 40A, the outer vessel 4002 has a number ofports 4020 a-4020 c (collectively 4020) to provide fluid communicationsor a fluid path between the outer chamber 4004 and an exterior of theouter vessel 4002. Three ports 4020 a-4020 c are shown in theillustrated embodiment, although fewer or greater number of ports may beemployed. Likewise, the inner vessel 4006 has a number of ports 4022a-4022 c (collectively 4022) to provide fluid communications or a fluidpath between the inner chamber 4008 and an exterior of the inner vessel4006. Three ports 4022 a-4022 c are shown in the illustrated embodiment,although fewer or greater number of ports may be employed. The ports4020, 4022 arranged such that selected ports on the inner vessel 4006align with selected ports on the outer vessel 4002 in a firstorientation or configuration (FIG. 40A), while selected ports on theinner vessel 4006 align with selected ports on the outer vessel 4002 ina second orientation or configuration, different from the firstorientation or configuration. The fluid path can be modified by simplyorienting the inner vessel 4006 with respect to the outer vessel 4002.For example, in the illustrated embodiment, a first fluid path betweenports 4020 a, 4020 b of the outer vessel 4002 is established when theinner vessel 4006 is in a first orientation (FIG. 40A) with respect tothe outer vessel 4002. Rotating the inner vessel 4006 with respect tothe outer vessel 4002, for instance 180 degrees about a longitudinalaxis, establishes a second fluid path between ports 4020 a, 4020 c ofthe outer vessel 4002. Notably, the inner wall or periphery that formsthe outer chamber 4004 selectively seals one of the ports 4022 a, 4022 cof the inner vessel 4006 depending on the orientation of the innervessel 4006 with respect to the outer vessel 4002. Other embodiments mayemploy additional ports. Ports may be oriented at angles other than 180degrees from one another. Thus, for example, the inner vessel 4006 mayhave ports oriented at 90 degrees, 60 degrees or 45 degrees from eachother. Such may provide a greater number of selectively selectable fluidpaths. While the outer and inner vessels 4002, 4006, respectively, areillustrated having an equal number of ports 4020, 4022, respectively, insome embodiments the number of ports of the outer and inner vessels4002, 4006 may not be equal to one another.

While the embodiments of FIGS. 38A-38B, 39A-39C, 40A-40B may all bemanually operated (e.g., manually rotated to select a desired flow pathor port), some embodiments may be automatically operated, for exampledrive by an electric motor, solenoid or other electrical orelectromechanical actuator.

FIG. 41 shows a method 4100 of isolating nucleic acid from a specimenaccording to one illustrated embodiment. Method 4100 may be useful inaspects of methods of lysing, extracting, capturing and isolatingbiological materials described elsewhere herein. Alternatively, method4100 may be useful in combination with a variety of other approaches tothe isolation of biological materials, particularly nucleic acids suchas DNA, known and practiced in the art.

At 4102, a specimen containing nucleic acid is obtained. The specimenmay include any of a variety of preparations containing nucleic acid forfurther processing. Such a specimen may range from relatively crudepreparations of lysed cells to relatively clean solutions of partiallypurified nucleic acid.

At 4104, a particulate material having an affinity for nucleic acid isobtained. The particulate material may have the form of a bead. Suchparticulate materials may include any of a variety of such materialsknown in the art and available for binding nucleic acids, including butnot necessarily limited to particles made from ceramic, glass, zirconia,silica, or sand, as well as particles having a metal core coated by amaterial that facilitates binding of nucleic acid.

At 4106, a low ionic strength zwitterion-containing buffer is obtained.A zwitterion is a polar chemical compound that at a particular pH,termed its isoelectric point (pI), has a positive and a negative chargeon different atoms and thus a net charge of zero. The acidic and basicfunctional groups of a zwitterion have dissociation constants, thelogarithms of which are termed, respectively, pKa and pKb (or pKa₁ andpKa₂). Within a range around these dissociation constants, zwitterionsserve as effective buffering agents. For example, several amino acidsare useful as buffering agent in a range between about pH 2 and pH 4. Atthe upper end of that range as the pH approaches the isoelectric pointthe net charge on the molecule decreases. Thus, for example, at pH 4 anamino acid such as glycine may serve as a buffer while contributingrelatively little to ionic strength of the solution. Many other aminoacids, aminosulfonic acids and aminocarboxylic acids may have similarutility as buffers while contributing relatively little to ionicstrength within a range near their pKa's.

At 4108, the specimen is contacted with the particulate material toallow at least a portion of the nucleic acid to bind to the particulatematerial. Composition of the specimen may help to induce binding of thespecimen to a particulate material. In methods commonly known in theart, nucleic acid-containing compositions at very high ionic strengthand pH near neutrality, such as conditions used during chemical lysis ofcells, promote binding of nucleic acids to surfaces such as varioustypes of silica, zirconia or ceramic surfaces. For example, commonlyused compositions that promote binding of nucleic acids to such surfacesmay include salts in a range of concentrations between about 2M andabout 6M, chaotropic chemical lysing agents such as guanidinehydrochloride or guanidine thiocyanate in a range as high as between 6Mand 10M, typically in a pH range between about 6 and about 8. Carryingout such operations requires further manipulation of the particle-boundnucleic acids to remove such materials in order to provide nucleic acidsthat are suitable for subsequent reaction and/or analysis, for example,amplification by PCR. Removing the salts and/or chaotropes requires, atminimum, extensive washing, typically also at a pH between about 6 andabout 8, often including alcohol as well. During wash, as the ionicstrength decreases at pH 6-8, the nucleic acids begin to release fromthe surface of the particulate, leading to decreased yields. Further,use of alcohol as a wash medium requires drying of the washed particlesto remove the alcohol. It has been surprisingly discovered that bysuitable selection of a buffering agent and a pH, particle-bound nucleicacids can be washed under conditions such that the nucleic acids remainbound to the particles. The nucleic acids can then be selectively elutedonce the wash is complete.

At 4110, the particulate material having bound nucleic acid is washedwith a zwitterion-containing buffer at a low pH and a low ionicstrength. For example, at pH 4 and low ionic strength nucleic acidremains bound to the silica, zirconia or ceramic particles. Suitablebuffer ions for use under such conditions to provide buffering at pH 4while contributing only low ionic strength to the solution include, asnoted above, various amino acids, aminosulfonic acids andaminocarboxylic acids. For example, low ionic strength glycine buffer atpH 4 is particularly suitable as a wash buffer for particle-boundnucleic acid. In certain embodiments, such buffer may be advantageouslyused to wash particle-bound nucleic acids following use of harshchemical lysing. Under such conditions the harsh agents are removedwhile the nucleic acids remain bound. Following washing with suchzwitterionic buffers at low ionic strength and low pH, the nucleic acidmay be readily released from the particles simply by increasing the pHto around neutrality, thus yielding a nucleic acid solution particularlysuitable for subsequent analysis, e.g., by PCR.

In certain embodiments, a zwitterion-containing wash buffer as describedabove may be used as a binding buffer because it not only preventsrelease but also induces binding of nucleic acids to particles under theconditions described.

Zwitterionic substances suitable for use in a wash buffer or a bindingbuffer as described may advantageously have pKa, i.e., pKa₁, valuesbetween about 2 and about 4.

In one embodiment, formulations suitable for carrying out the methodsdescribed herein, including formulations for washing particle boundnucleic acids, may include formulations having a low ionic strengthzwitterion-containing buffer having a pH between about 3.5 and about 5and including zwitterionic substances with a pKa between about 2 andabout 4 and/or a pKb between about 9 and about 11.

In certain embodiments, a kit may be suitable for use in methods forisolating a nucleic acid. In certain embodiments, a kit may include aparticulate material that has affinity for nucleic acid and a low ionicstrength zwitterionic buffer having a pH less than about 6. In certainembodiments, the kit may include instructions for using the kit contentsto isolate a nucleic acid. In certain embodiments, the low ionicstrength zwitterion-containing buffer may have a pH between about 3 andabout 6. In certain embodiments, the zwitterionic buffer in the kit mayinclude an amino acid, an aminosulfonic acid, or an aminocarboxylicacid.

FIG. 42 shows a method 4200 for carrying out a particular aspect ofmethod 4100 directed to isolating a nucleic acid in one illustratedembodiment.

At 4202, a specimen is mixed with buffer to adjust the pH of thespecimen before contacting the specimen with a particulate material. Ina certain embodiment, the pH of the specimen may be adjusted to below 5to induce binding of nucleic acid from the specimen to the particulatematerial.

FIG. 43 shows a method 4300 for carrying out a particular aspect ofmethod 4100 directed to isolating a nucleic acid in one illustratedembodiment.

At 4302, a specimen is mixed with a chaotropic agent before contact withthe particulate material. In a certain embodiment, the chaotropic agentmay be used at a concentration sufficient to chemically lyse abiological material in the specimen. For example, the chaotropic agentmay lyse a cell wall. In a certain embodiment, the chaotropic agent mayinduce binding of nucleic acid to the particulate material. In method4100, washing particles having bound nucleic acid with low ionicstrength, low pH zwitterionic buffer allows efficient removal of thechaotropic agents without dissociating the nucleic acid from theparticulate material.

FIG. 44 shows a method 4400 for carrying out a particular aspect ofmethod 4100 directed to isolating a nucleic acid in one illustratedembodiment.

At 4402, a specimen is mixed with ionic compounds to adjust theconcentration of ionic compounds in the specimen. In certainembodiments, ionic compounds may be added to adjust the concentration tobetween 2M and 6M. High concentrations of ionic compounds may be used toinduce binding of nucleic acid to particulate material. In method 4100,washing particles having bound nucleic acid with low ionic strength, lowpH zwitterionic buffer allows efficient removal of the highconcentrations of ionic compounds that may be used initially to inducebinding of the nucleic acid to the particulate material.

FIG. 45 shows a method 4500 for further processing the particulatematerial having bound nucleic acid after washing with thezwitterion-containing buffer in one illustrated embodiment.

At 4502, a low ionic strength buffer having a pH above 6 is applied tothe washed nucleic acid-containing particulate material. Applying thebuffer increases the pH at the surface of the particulate material, thusneutralizing residual wash buffer and eluting the bound nucleic acidwhile maintaining a low ionic strength.

FIG. 46 shows a method 4600 for further processing nucleic acid elutedfrom the particular by method 4500 in one illustrated embodiment.

At 4602, the eluted nucleic acid is provided for further processing inan amplification reaction, e.g., by PCR. Nucleic acid in a low ionicstrength buffer at a pH near neutrality as obtained from methods 4100and 4500 is particularly suitable for further processing and analysis.

FIG. 47 shows a method 4700 for isolating phosphate-containingpolyanions using an amine-containing solid phase material in oneillustrated embodiment. Phosphate-containing polyanions include nucleicacids such as DNA.

At 4702, a specimen having phosphate-containing polyanions is obtained.At a pH of around about 7, the phosphate-containing polyanions have anegative charge. Such polymeric molecules may be obtained from cellsthat are either mechanically or chemically lysed. Cells that aremechanically lysed to obtain phosphate-containing polyanions may belysed by methods described elsewhere herein.

At 4704, an amine-containing or aminated solid phase material isobtained. At a pH of around about 7, the amine groups on theamine-containing solid phase material are positively charged. Thus, thepositively charged amine-containing solid phase material will bind thenegatively charged phosphate-containing polyanions. However, thepositively charged amine-containing solid phase material may also bindother negatively charged molecules that may be present in the specimen.Thus eluting phosphate-containing polyanions from the amine-containingsolid phase material may elute not only the polyanions of interest butalso other negative charge species. Accordingly, such would notnecessarily be a reliable approach to isolating phosphate-containingpolyanions, e.g., nucleic acids such as DNA. An alternative approachtakes advantage of the phosphate-containing moieties in the polyanionand the possible involvement of such moieties in the binding of thepolyanion to the amine-containing solid phase material.

At 4706, a formulation of phosphate-containing anions is obtained. Theformulation may include one or more of a variety of phosphate-containinganions. For example, such anions may include organic or inorganicphosphates. Phosphate-containing anions may be considered and selectedon the basis of possible chemical and/or structural similarity to thephosphate moieties on the phosphate-containing polyanions.

At 4708, the specimen containing the phosphate-containing polyanion iscontacted with the amine-containing solid phase material to allow thephosphate-containing polyanion to bind to the amine-containing solidphase material.

FIG. 48 shows a method 4800 for eluting the phosphate-containingpolyanion from the amine-containing solid phase material in oneillustrated embodiment.

A positively charged surface such as an amine-modified solid phasematerial, e.g., silica, may bind a polyanion such as aphosphate-containing polyanion. In particular, such a positively chargedsurface may bind nucleic acid. Typically releasing aphosphate-containing polyanion bound to such a positively chargedsurface would require ion exchange. However, elution ofphosphate-containing polyanions may be accomplished more efficient byselecting and using appropriate anions.

At 4802, the formulation of phosphate-containing anions is applied tothe amine-containing solid phase material to which thephosphate-containing polyanion is bound.

Applying the phosphate-containing anion elutes the phosphate-containingpolyanion. The phosphate-containing anionic formulation used to elutethe phosphate-containing polyanion may include any phosphate anionhaving a structure suitable to compete with the phosphate moieties onthe polyanion for binding to the amine-containing solid phase.

In a certain embodiments, the phosphate-containing polyanion is anucleic acid, including DNA and RNA. While phosphate-containing anionsin general may elute phosphate-containing polyanions from theamine-containing solid phase materials, use of nucleoside triphosphatesmay improve efficiency of release of nucleic acids by as much asten-fold. Elution by nucleoside triphosphates may leave other polyanionslacking phosphate bound to the amine-containing solid phase material.This approach may be particularly useful when isolating nucleic acidsfrom natural materials that have a high load of anionic proteins,anionic polysaccharides, or other polyanions. Nucleoside triphosphatessuitable for elution of nucleic acids from the aminated surfaces mayparticularly include at least deoxyadenosine triphosphate,deoxyguanosine triphosphate, deoxycytidine triphosphate, anddeoxythymidine triphosphate, adenosine triphosphate, guanosinetriphosphate, cytidine triphosphate, and uridine triphosphate.

In certain embodiments, a kit may be suitable for use in methods forisolating. In certain embodiments, a kit may include an amine-containingsolid phase material that has affinity for phosphate-containingpolyanions such as nucleic acids. In certain embodiments, the kit mayinclude instructions for using the kit contents to isolatephosphate-containing polyanions.

FIG. 49 shows a method 4900 for isolating sulfonate-containingpolyanions using an amine-containing solid phase material in oneillustrated embodiment.

As discussed above, amine-containing solid phase materials may be usedto isolate phosphate-containing polyanions such as nucleic acids.Natural materials may contain sulfonated polyanions in addition to thephosphate-containing polyanions. The negatively charged sulfonatedpolyanions will also bind to the positively charged amine-containingsolid phase. In certain embodiments, it may be advantageous to removebound sulfonated polyanions before isolating the phosphate-containingpolyanions.

At 4902, a specimen having sulfonate-containing polyanions is obtained.In certain embodiments, this specimen may likely also havephosphate-containing polyanions. At a pH of around about 7, thesulfonate-containing polyanions have a negative charge. If present,phosphate-containing polyanions will also have a negative charge.

At 4904, an amine-containing or aminated solid phase material isobtained. At a pH of around about 7, the amine groups on theamine-containing solid phase material are positively charged. Thus, thepositively charged amine-containing solid phase material will bind thenegatively charged species in the specimen, includingsulfonate-containing polyanions, as well as any phosphate-containingpolyanions present in the specimen.

At 4906, a formulation of sulfate-containing anions is obtained. Theformulation may include one or more of a variety of sulfate-containinganions. For example, such anions may include organic or inorganicsulfates.

At 4908, the specimen containing the sulfonate-containing polyanion iscontacted with the amine-containing solid phase material to allow thesulfonate-containing polyanion to bind to the amine-containing solidphase material.

FIG. 50 shows a method 5000 for eluting the sulfonate-containingpolyanion from the amine-containing solid phase material in oneillustrated embodiment.

At 5002, the formulation of sulfate-containing anions is applied to theamine-containing solid phase material to which the sulfonate-containingpolyanion is bound. Applying the sulfate-containing anion elutes thesulfonate-containing polyanion. The sulfate-containing anionicformulation used to elute the sulfonate-containing polyanion may includeany sulfate anion having a structure suitable to compete with thesulfonate moieties on the polyanion for binding to the amine-containingsolid phase.

Upon removal of the sulfonate-containing polyanion, phosphate-containinganions bound to the amine-containing solid phase may be advantageouslyeluted with a lower possibility of contamination of the isolatedphosphate-containing polyanions by sulfate-containing polyanions.

FIG. 51 shows method 5100 for capturing microorganisms having high cellwall lipid content on a material having a hydrophobic surface accordingto one illustrated embodiment. Method 5100 may be useful in aspects ofmethods of lysing, extracting, capturing and isolating biologicalmaterials described elsewhere herein. Alternatively, method 5100 may beuseful in combination with other approaches to the isolation ofmaterials known and practiced in the art.

The outer surface of cell walls of microorganisms with high cell walllipid content may include both hydrophobic and charge characteristics.For example, mycobacterial cell walls have both high lipid content and anet negative charge. Thus, mycobacteria are attracted to hydrophobicsurfaces as well as to surfaces that have a positive charge. Theinterest in capturing microorganisms with high cell wall lipid content,particularly mycobacteria, is to isolate and further process DNA fromthese microorganisms once captured. However, various approaches tocapturing such microorganisms present certain challenges. Solutions tosuch challenges are discussed below.

At 5102, a surface of a solid phase material is modified to make thesurface hydrophobic in order to induce binding of a microorganism havinga high cell wall lipid content to the surface. In this embodiment, thesurface has no net charge.

At 5104, a specimen containing microorganisms having high cell walllipid content contacts the hydrophobic surface of the solid phasematerial.

At 5106, the hydrophobic surface of the material captures microorganismshaving high cell wall lipid content. In one embodiment, silica particlesmodified to have a hydrophobic surface with no charge were shown to bindMycobacteria bovis cells from a suspension at high efficiency.

FIG. 52 shows a method 5200 of lysing in a suitable mediummicroorganisms captured on a hydrophobic surface in one illustratedembodiment.

At 5202, a medium having low-to-moderate ionic strength is supplied to asolid phase material having a hydrophobic surface to which is boundmicroorganisms with high cell wall lipid content.

At 5204, the bound microorganisms with high cell wall lipid content arelysed. At low-to-moderate ionic strength, DNA released from the cell isnot bound to the hydrophobic surface of the solid phase material. TheDNA is thus in a medium suitable for subsequent processing and analysis.

FIG. 53 shows a method 5300 of amplifying DNA in a lysate from amicroorganism with high cell wall lipid content, e.g., by PCR, in oneillustrated embodiment.

At 5302, DNA in a lysate from microorganisms having high cell wall lipidcontent is amplified, e.g., by PCR. The lysate is from microorganismscaptured on a hydrophobic solid phase and lysed.

FIG. 54 shows method 5400 for capturing microorganisms having high cellwall lipid content on a solid phase material having a hydrophobic andpositively charged surface according to one illustrated embodiment.Method 5400 may be useful in aspects of methods of lysing, extracting,capturing and isolating biological materials described elsewhere herein.Alternatively, method 5400 may be useful in combination with otherapproaches to the isolation of materials known and practiced in the art.

At 5402, a surface of a solid phase material is modified to make thesurface hydrophobic and positively charged in order to induce binding ofa microorganism having a high cell wall lipid content to the surface.Surface modifications include reaction with polydiallyldimethylammoniumchloride and other surface modifications to confer hydrophobicity andpositive charge on the surface of the solid phase material. In anotherembodiment, the surface of the solid phase may be modified with analkylamine compound. For example, silica bead surfaces may be modifiedwith 3-aminopropyl silane. Such beads can effectively capture bothMycobacteria bovis and Clostridium cells from a sample.

At 5404, a specimen containing microorganisms having high cell walllipid content contacts the hydrophobic and positively charged surface ofthe solid phase material.

At 5406, the hydrophobic surface of the material captures microorganismshaving high cell wall lipid content.

FIG. 69 shows several preferred embodiments of solid phase beadsmodified with different molecules which may be used to bind to cells ofdifferent types.

FIG. 55 shows a method 5500 of lysing in a suitable mediummicroorganisms captured on a hydrophobic surface in one illustratedembodiment.

At 5502, a medium having deoxynucleoside triphosphates is supplied to asolid phase material having a hydrophobic and positively charged surfaceto which is bound microorganisms with high cell wall lipid content.

At 5504, the bound microorganisms with high cell wall lipid content arelysed. Deoxynucleoside triphosphate in the lysing medium competitivelyinhibits DNA binding to the surface of the solid phase material.Deoxynucleoside triphosphates in the lysate do not interfere withsubsequent amplification reactions. The DNA is thus in a medium suitablefor subsequent processing and analysis.

FIG. 56 shows a method 5600 of amplifying DNA in a lysate from amicroorganism with high cell wall lipid content, e.g., by PCR, in oneillustrated embodiment.

At 5602, DNA in a lysate from microorganisms having high cell wall lipidcontent is amplified, e.g., by PCR. The lysate is from microorganismscaptured on a hydrophobic and positively charged solid phase and lysed.

FIG. 57 shows method 5700 for capturing microorganisms having high cellwall lipid content on a solid phase material having a hydrophobic andnegatively charged surface according to one illustrated embodiment.Method 5400 may be useful in aspects of methods of lysing, extracting,capturing and isolating biological materials described elsewhere herein.Alternatively, method 5700 may be useful in combination with otherapproaches to the isolation of materials known and practiced in the art.

At 5702, a surface of a solid phase material is modified to make thesurface hydrophobic and negatively charged in order to induce binding ofa microorganism having a high cell wall lipid content to the surfaceunder appropriate conditions.

At 5704, a specimen containing microorganisms having high cell walllipid content contacts the hydrophobic and negatively charged surface ofthe solid phase material.

At 5706, the hydrophobic and negatively charged surface of the materialcaptures microorganisms having high cell wall lipid content. Thespecimen is in a medium having high salt concentration and/or low pH.Under these conditions binding of the hydrophobic and negatively chargedsurface of the microorganism to the hydrophobic and negatively chargedsurface of the solid phase material is aided by bridging of thenegatively charged groups via cations in solution as a result of thehigh salt concentration and/or the low pH.

FIG. 58 shows a method 5800 of lysing in a low ionic strength mediummicroorganisms captured on a hydrophobic and negatively charged surfaceof the solid phase material in one illustrated embodiment.

At 5802, a medium having low ionic strength is supplied to a solid phasematerial having a hydrophobic and negatively charged surface to which isbound microorganisms with high cell wall lipid content.

At 5804, the bound microorganisms with high cell wall lipid content arelysed. The low ionic strength during lysis inhibits binding of nucleicacid to the surface of the solid phase material and provides a mediumsuitable for subsequent amplification reactions, e.g., PCR.

FIG. 59 shows a method 5900 of amplifying DNA in a lysate from amicroorganism with high cell wall lipid content, e.g., by PCR, in oneillustrated embodiment.

At 5902, DNA in a lysate from microorganisms having high cell wall lipidcontent is amplified, e.g., by PCR. The lysate is from microorganismscaptured on a hydrophobic and negatively charged solid phase and lysed.

In methods 5100 to 5900 the surfaces of the solid phase materialincludes surfaces of particulates, beads, membranes, flow channels andtubes, any of which may act to capture cells with high lipid cell wallcontent for subsequent lysis and release of nucleic acids.

Mycobacterium tuberculosis has cell walls that are over 60% lipid.Mycolic acids form a lipid shell around the cell. The cell walls includethree main types of mycolic acids: alpha-, methoxy-, and keto-. Alphamycolic acids comprise at least 70% of the mycolic acids present in theorganism. The lipid chemistry of M. tuberculosis cell walls provides thebasis for binding of the species to surfaces that are coated withdiallyldimethylammonium chloride. Similar binding can be seen on silicabeads that have been modified by reaction with various silanes to conferhydrophobicity or a combination of hydrophobicity and positive charge tothe surface of the beads. The siloxol component of the silanesfacilitates polymerization of monomers and coupling to a silica surfaceor other mineral oxide surfaces. There are many alkane and vinyl silanesthat can confer hydrophobicity to the silica surface and there are manyorganic amino silanes that may be combined to achieve an optimum ratioof the two characteristics of hydrophobicity and charge that maximizeaffinity of Mycobacteria. In certain embodiments, surfaces that are atleast primarily hydrophobic may be advantageous for the processing ofnucleic acids, as such will limit the potential for the surfaces to bindnucleic acids once they have been released from cells.

FIG. 60 shows a method 6000 of obtaining a biological material ofinterest from a specimen in one illustrated embodiment.

At 6002, a specimen having a mixture of different species of biologicalmaterials is introduced into a chamber containing mixed population ofparticulate material having different sizes and different bindingaffinities for the different species of biological materials in thespecimen.

At 6004, the specimen is lysed by agitating the specimen in the chamberwith the mixed population of particulate material and a fluid.

FIG. 61 shows a method 6100 of obtaining a biological material ofinterest from a specimen in one illustrated embodiment.

At 6102, a specimen having a mixture of different species of biologicalmaterials is introduced into a chamber containing particulate lysingmaterial having an affinity for the biological material of interest andparticulate secondary binding material having affinity for one or moresecondary biological materials other than the material of interest. Theparticulate secondary binding material is of a size smaller than theparticulate lysing material. The particulate lysing material and theparticulate secondary binding material may each include a plurality ofbeads. The particulate lysing material may include at least one of aplurality of ceramic beads, a plurality of glass beads, a plurality ofzirconia beads, a plurality of silica beads, a plurality of sand, or aplurality of beads with a metal core coated by a material thatfacilitates binding of the biological material of interest. Theparticulate secondary binding material may include at least one of aplurality of ceramic beads, a plurality of glass beads, a plurality ofzirconia beads, a plurality of silica beads, a plurality of sand, or aplurality of beads with a metal core coated by a material thatfacilitates binding of the biological material of interest. Thebiological material of interest may be a nucleic acid. Secondarybiological materials may include materials that interfere with bindingthe biological material of interest by the particulate secondary bindingmaterial or materials that interfere with subsequent reactions with oranalysis of the biological material of interest. The particulatesecondary binding material may be include paramagnetic particle.Paramagnetic beads may be sequestered, thus providing a means forinterfering reagents to be sequestered from the lysate during lysis ofspecimens or providing a means for subsequent extraction and analysis ofa secondary analyte. The secondary binding material may include proteinA, protein G, protein L, anti-albumin antibody, concanavalin A ortitanium dioxide.

At 6104, the specimen is lysed by agitating the specimen in the chamberwith the particulate lysing material, the particulate secondary bindingmaterial, and a fluid.

FIG. 62 shows a method 6200 of obtaining a biological material ofinterest that includes further aspect of method 6100, in one illustratedembodiment.

At 6202, a filter is selected having passages of a size to substantiallypass the particulate secondary binding material and to substantiallyprevent passage of the particulate lysing material.

At 6204, a mixture of lysed specimen combined with the mixed populationof particulate matter and fluid is flowed through the filter.

In one embodiment, the particulate lysing material binds the biologicalmaterial of interest and the particulate secondary binding materialbinds secondary biological material(s) that are not of interest or thatmay interfere with binding or subsequent reactions of the biologicalmaterial of interest. By binding secondary biological material(s) to theparticulate secondary binding material and then passing it through afilter that retains the particulate lysing material, the secondarybinding material(s) may be physically removed from the biologicalmaterial of interest and discarded. Alternatively, the secondarybiological material(s) may be retained for some other use. The smallersize of the particulate secondary binding materials not only allowstheir separation from the larger particulate lysing material but alsogives them greater surface area and thus greater binding capacity perunit mass. Although the particulate secondary binder materials are smallrelative to the particulate lysing materials, they are large enough andhave sufficient inertia to prevent their binding to the particulatelysing material during agitation and lysis of the specimen. Low bindingbeads (OPS Diagnostics, Lebanon, N.J.) are commercially available silicaor zirconia beads that have been overcoated. Such beads are hydrophobicand have a low binding affinity for DNA. Such beads have been tested fortheir ability to bind biological materials, such as lipids or proteins,which may interfere with binding of nucleic acids to zirconia beads. Incertain embodiments, particulate lysing materials may be beads having adiameter of 50-250 μm. These beads may be retained by a filter or mesh.Using particulate secondary binding beads smaller than the pore size ofthe filter or mesh allows them to be discarded with the lysate.

FIG. 63 shows a method 6300 that includes a further aspect of methods6000 to 6200 in one illustrated embodiment.

In 6302, a particulate material retained on the filter is recovered.

In 6304, biological material is eluted from the particular materialretained on the filter.

FIG. 64 shows a method 6400 for rapidly processing and capturing abiological material in one illustrated embodiment.

At 6402, a device having a chamber containing a solid phase material andan attached fitting holding a thin filter insert is obtained. The poreor mesh size of the filter and the thickness are suitable to allow rapidflow at low pressure. Elements to retain particulate materials in suchdevice typically limit flow through the device. In one embodiment, thedevice has a filter or mesh having a thickness of 0.002″-0.003″. Deviceswith thicker such elements often provide slow and typically requirepressure. Such retention elements may often include frits, such as glassfits, or dense filters.

Rapid flow and low pressure are advantageous in that procedures can becompleted in less time, pumps and fluidic systems are not required toproduce high pressure, and fluidic components which cannot withstandhigh pressure may be used. By rapid flow or substantially uninhibitedflow or uninhibited flow is meant a flow rate of greater than about 0.1ml/min and, more preferably, greater than about 1 ml/min. By lowpressure or low operating pressure is meant a pressure less than about200 psi and, more preferably less than about 100 psi. Such pressures toinduce flow can readily be applied with a syringe, pipettor, peristalticpump, or syringe pump.

At 6404, a fluid specimen containing a biological material is introducedinto the chamber of the device to bind to the solid phase material.

At 6406, the fluid specimen flows through the solid phase material,exiting the device through the filter insert. The device with the filterinsert allows rapid flow.

FIG. 65 shows a method 6500 of recovering biological material in oneillustrated embodiment.

At 6502, biological material is eluted from a solid phase materialretained in a chamber of a device by a thin filter or wire mesh. Elutionmay be carried out at low pressure while allow rapid flow.

At 6504, the effluent is collected from the solid phase material exitingthe device through the thin filter or wire mesh.

In one embodiment, proteins or peptides may be captured by solid phasematerial contained in a chamber of a device that allows high flow rateand low pressure in comparison to other devices known in the art. Incertain embodiments, the solid phase material may be in the form of aparticulate or bead. In certain embodiments, such a device may have athin filter or mesh material.

In certain embodiments, the chamber of such a device may contain solidparticulates having physical characteristics such that they do not packas densely as certain materials known in the art, such as agarose beadsor silica gel. In certain such embodiments, fluids may flow around theparticulates relatively unimpeded yet may have sufficient opportunity tointeract with the surfaces of the particulates to allow proteins orother biomolecules or biological materials to interact with bindingligands on the surfaces of the particulates. The particulates maycomprise materials which may be surface-modified with capture ligands tobind proteins or other biomolecules or biological materials. In certainembodiments, particulates may include metal oxides, ceramics, or othersuch relatively non-compressible materials. In certain such embodiments,particulates may include silicon dioxide, zirconia dioxide, or titaniumdioxide. In other embodiments, particulates may include silica gel,sepharose, or agarose. Such particulate materials may pack more denselythan metal oxides, ceramics, and the like. Such relatively compressibleparticulate materials may impede flow of fluid through such a device.The particulate material may also comprise mixtures of more than onematerial such as a mixture of silicon dioxide and zirconia dioxide. Incertain embodiments, particulates may have a spherical or anapproximately spherical shape. In other embodiments, particles may havean irregular or random shape, such as that of crushed silica or glassshards.

The particulates may be of any size. Size is generally selected toprovide a bed of particles that allows a sufficiently high flow rate atlow pressure. Generally, larger particulates tend to allow flow at lowpressure that is greater than that allowed by smaller particulates. Onthe other hand, smaller particulates have larger surface area and thushigher binding capacity and more rapid binding kinetics than largerparticulates. Particle size is thus chosen to optimize operationalcharacteristics, that is, to achieve high flow rate at low pressure andto provide sufficient binding capacity and kinetics to allow adequateprotein or biomolecule capture. The device includes a filter or meshmaterial to trap the particulates in the chamber. The filter or meshmaterial has pores or openings selected to preclude passage of theparticulate material and also to allow rapid flow at low pressure. Priorart devices typically include frits, e.g., glass frits, to retain solidparticulates in chambers. Such devices tend to have slow flow rates, andthe pores in the frits also tend to clog during use. In certainembodiments of the device disclosed herein, the filter or mesh isselected to have a pore size and shape such that the particulatematerial does not readily clog the filter or mesh. For example,spherical particles may more easily clog round pores than square poresin the filter or mesh material.

The filter or mesh utilized in the device disclosed herein may be of anysolid material, including but not necessarily limited to plastic (e.g.,nylon, polyester, polyethylene, and Teflon), metal, or ceramic. Incertain embodiments, the filter or mesh material may be stainless steel.Filter or mesh materials may be solid materials having holes or pores ormay be woven from strands of the material. In a woven filter or mesh,the weave pattern may have any of a variety of patterns known in theart. Different weave patterns may be chosen, for example, to achievedifferent pore size and geometry to achieve adequate retention of theparticulate material in the chamber while allowing high flow rate at lowpressures.

In certain embodiments, the particulate material may comprise gold.Solid gold particles or particles of a different material but coatedwith gold may be used. Gold particles can readily be surface-modifiedwith a variety of ligands of different chemical structures, for example,by binding the ligands using thiol monolayer technology as is well knownin the art.

Many particulate materials are known to bind nucleic acids withoutsurface chemical modification. Silica materials are widely used in theart for such purposes. Particulate materials for use in the devicesdisclosed herein may have surfaces that are modified to bind particularbiological materials. Such surface modifications may be carried out by avariety of methods well known in the art. Such modifications may achieveimmobilization of binding ligands on the surfaces of the particles. Suchbinding ligands may be chosen to selectively bind particular biologicalmaterials. Without limitation, the following ligand binding pairs may beincorporated on the surface of the particulates to capture proteinsand/or other biological molecules or materials.

Surface-bound ligand Binding Partner Nickel chelates His-tagged proteinor peptide Glutathione GST-tagged protein or peptide Biotin Streptavidin(SA) and SA-labeled protein and peptide Avidin, streptavidin andBiotin-labeled protein and peptide analogs Streptavidin-bindingStreptavidin (SA) and SA-labeled protein and peptide peptideCalmodulin-binding Calmodulin protein Antibodies Antigens: Smallmolecules, peptides, proteins, nucleic acids, polysaccharides AntigensAntibodies Protein G Immunoglobulins Protein A Immunoglobulins Protein LImmunoglobulins Cibachron Blue Albumin Sulfate Albumin Intercalators(e.g., Double-stranded DNA, RNA/DNA ethidium bromide) heteroduplexesSybr Green I Double-stranded DNA Sybr Green II Single-stranded DNA & RNAMaltose Maltose binding protein 9-mer peptide FLAG (DYKDDDDK) 10-merpeptide MYC (EQKLISEEDL) Concanavalin A Glycoproteins and carbohydratesBenzylguanine SNAP-tag (20 kDa mutant of the human DNA repair proteinO⁶-alkylguanine-DNA alkyltransferase (hAGT) Enzyme inhibitor Enzyme

Additional binding pairs are well known in the art. Affinity tags forprotein purification are reviewed in Lichty, et al. (Lichty, J. J., etal., Protein Expr. Purif., 41(1):98-105, 2005), the entirety of which isincorporated herein by reference.

In certain embodiments the surface-bound ligand and the binding partnerbecome bound covalently. For example, when the surface-bound ligand isan enzyme suicide inhibitor, a covalent bond is often formed when theenzyme binds the inhibitor. Similarly, when the SNAP-tag binds tobenzylguanine, the SNAP-tag enzyme becomes covalently coupled. In suchcases, a linker can optionally be used to tether the surface-boundligand to the surface which contains a cleavable group. In this case,the captured protein of peptide can be eluted by cleaving the cleavablegroup. Examples of cleavable groups include disulfides, which can becleaved, for example, by dithiothreitol and ester bonds which can becleaved by acids (low pH). Cleavable groups can also be photolabile. Forexample, substituted 3-nitro-2-naphthalenemethanol groups can beincorporated into the linker and readily cleaved to allow elution using380 nm wavelength electromagnetic radiation (light).

In another embodiment the surface bound ligand may be attached to thesurface of beads by a linker comprising oligonucleotides. Oneoligonucleotide having a specific sequence may be attached to thesurface of the bead and a second oligonucleotide which is at leastpartially complementary to the first oligonucleotide may be attached tothe ligand. Under the proper conditions of temperature, pH, and solutionchemistry the two oligonucleotides may bind to one another andeffectively link the ligand to the surface. In this embodiment,following binding of the protein, nucleic acid, or peptide to the beadsurfaces, the bound molecules can be eluted by subjecting the beads toconditions such as low ionic strength or high temperature, which causethe two oligonucleotides to dehybridize or melt.

In certain embodiments particulate material may be modified to provideat its surface a positive charge (e.g., by surface modification withamine-containing moieties). In certain such embodiments, particulatematerials may be used to bind to negatively charged proteins or otherbiomolecules or biological materials. In other embodiments, the surfacemay be modified to have negatively charged groups (e.g., carboxylicacids, sulfates, or phosphates). In certain such embodiments,particulate materials may be used to bind to positively charged proteinsor other biomolecules or biological materials.

In certain embodiments, particulate material may be modified to containpores in its surface. In certain such embodiments, particulate materialmay be used to segregate small and large molecules (size with respect tothe pore size). Thus, for example, such pore-containing particulates ina chamber may be used to retain small-sized biomolecules and allowlarge-sized biomolecules to pass through the chamber.

FIG. 68 shows a device 6800 for capturing a protein or other biomoleculeor biological material from a fluid specimen at a high flow rate and alow operating pressure according to one illustrated embodiment.

In this one illustrated embodiment, the particulate material iscontained in a chamber or cartridge 6808 having an entrance port 6816and an exit port 6818. A filter or mesh material 6810 is positioned atthe exit port 6818 and optionally a filter or mesh material 6806 ispositioned at the entrance port 6816 to retain the particulate materialin the chamber 6808. The chamber 6808 may be in the form of a cylinderor any other shape which facilitates high flow rate and low pressureflow of fluids through the chamber and achieves approximatelyhomogeneous flow around the particulates. Device 6800 further includesinlet cap 6804 with inlet fitting 6802 and outlet cap 6812 with outletfitting 6814. Inlet cap 6804 is inserted into entrance port 6816 ofchamber 6808. Outlet cap 6812 is inserted into outlet port 6818 ofchamber 6808.

In certain embodiments, the chamber of the device 6800 for capturing aprotein or other biomolecule or biological material may be the samechamber containing a micromotor and an impeller used for lysing cellsand tissues as described elsewhere herein. In such embodiments, theparticulates used for lysing and the particulates used for capture ofproteins and/or other biomolecules on biological materials may be thesame. Alternatively, different particles may be employed to achievelysis and capture.

In certain embodiments, the chamber for capture of the protein or otherbiomolecules or biological materials may be separate from the deviceused for cell lysis. In certain such embodiments, the capture cartridgemay be placed downstream from the lysis cartridge. In certain other suchembodiments, the capture cartridge may optionally be placed upstreamfrom the lysis device.

In certain embodiment, binding cartridges may be placed both upstreamand downstream from the lysis cartridge. In certain such embodiments,the configuration may be advantageously used, for example, for removalof interfering substances by the upstream binding cartridge and captureof proteins or other biomolecules or biological materials by thedownstream cartridge.

In certain embodiments, cartridges or chambers of the device describedherein may be fluidly connected to one another by tubing. In suchembodiments, flow may thus occur from one cartridge to another. Incertain such embodiments, fluid may pass in one direction only. In othersuch embodiments, fluid may flow successively in opposite directionssuch that the fluid may pass through the cartridge(s) multiple times.

In one embodiment the chamber for capture of nucleic acids is separatefrom and downstream of the chamber for lysis. In this example the fluidsample is flushed through the lysis chamber and through the capturechamber multiple times. It was demonstrated that DNA and/or RNA from thelysed cells is effectively captured upon its first pass through thecapture chamber and does not pass through the lysis chamber onsubsequent flushing steps. It was shown that this procedure effectivelylimits any shearing of the DNA or RNA into smaller fragments.

In an alternative embodiment the sample is flushed multiple timesthrough the lysis chamber to effectively shear the DNA or RNA intosmaller fragments. It was shown that the degree to which the DNA or RNAis thus sheared can be affected and controlled, for example by thenumber of flushes through the chamber, the flow rate through thechamber, the speed of the motor, and the size of the lysing beads.

In the embodiments disclosed herein for capturing his-tagged proteins orpeptides, the particulate material is modified with a chelator bound tonickel. Examples of chelating groups include, without limitation,iminodiacetic acid (IDA), nitriloacetic acid (NTA), andtris(carboxymethyl)-ethylenediamine (TED).

In certain embodiments, following capture of the protein or otherbiomolecule or biological material, the particulate material mayoptionally be washed to remove unwanted chemicals and/or cellulardebris. In certain such embodiments, following the optional wash, thecaptured protein or other biomolecule or biological material may beeluted from the particulate material by flowing an elution bufferthrough the chamber. The elution buffer may comprise chemicals whichdisrupt the binding of the biomolecule or other biological material tothe surface-bound ligand. In certain embodiments, elution buffers mayadvantageously have a pH less than 6.0. In certain embodiments, thebuffer may include imidazole for eluting his-tagged protein or peptidefrom nickel chelate ligand. In other embodiments, the buffer may includeurea and have a low pH for eluting antibodies from antigen ligands andfor eluting immunoglobulin from protein G, protein L, or protein Aligands. Buffer solutions for eluting proteins and other biomolecules orbiological materials are well known in the art.

In further embodiments, when nickel chelators are employed to capturehis-tagged proteins or peptides, the elution buffer may includeethylenediaminetetraacetic acid (EDTA). In certain such embodiments, theEDTA may have a higher affinity for nickel than does the capture ligandand may thus effectively compete for nickel atoms and release thehis-tagged protein or peptide from the particulate material.

FIG. 66 shows a method 6600 for withdrawing samples forparticulate-containing specimens in one illustrated embodiment.

At 6602, a pipette tip having a filter or mesh material proximate an endof the pipette tip is attached to a pipette or syringe. The filter ormesh material may be inserted in the pipette tip or it may be insertedin a fitting that attaches to the pipette tip, as shown in FIGS. 67A and67B.

At 6604, the end of the pipette tip having the filter or mesh materialis inserted into a chamber or container holding a particulate-containingspecimen.

At 6606, fluid is drawn from the chamber or container into the pipettetip through the filter or mesh insert. This device may be used to removeparticulates from a specimen while withdrawing the specimen from acontainer or well. This device can be used, for example, to processsamples that have been lysed in a non-flow through mode. Aftercompletion of the lysis, the soluble materials can be withdrawn into thepipette tip while the particulates may be caught by the filter or mesh.

FIG. 67A shows one illustrated embodiment of a pipette tip for use inmethod 6600 in one illustrated embodiment. The filter or mesh materialis included within the pipette tip.

FIG. 67B shows another illustrated embodiment of a pipette tip for usein method 6600 in one illustrated embodiment. The filter or meshmaterial is included within a separate hold that may be affixed to astandard pipette tip before use.

The various embodiments described above can be combined to providefurther embodiments. U.S. provisional patent application Ser. No.61/020,072 filed Jan. 9, 2008; International Patent Application SerialNo. PCT/US2009/030622 filed Jan. 9, 2009 and published as WO2009/089466;U.S. provisional patent application Ser. No. 61/117,012 filed Nov. 21,2008; U.S. provisional patent application Ser. No. 61/220,984 filed Jun.26, 2009; U.S. nonprovisional patent application Ser. No. 12/732,070filed Mar. 25, 2010; U.S. provisional patent application Ser. No.61/317,604 filed Mar. 25, 2010; U.S. nonprovisional patent applicationSer. No. 12/823,081 filed Jun. 24, 2010; U.S. provisional patentapplication Ser. No. 61/427,045 filed Dec. 23, 2010; and U.S.provisional patent application Ser. No. 61/444,607 filed Feb. 18, 2011are incorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

The lysis and DNA extraction systems can be integrated with othercomponents and functions. This is especially true of pumps and valvesthat further enable integration of waste reservoirs, wash reservoirs andfunctions, chambers for sample or specimen introduction, elution bufferchambers and functions, chambers and functions for amplification anddetection. All of these structures and functions can be integrated intoa disposable container or cartridge that includes the bead beatingfunction for either lysis or analyte extraction or both. Integratingthese structures and functions into a disposable unit has practicaladvantages for point-of-care and point-of-use diagnostic applications.Integrating these structures functions into a disposable unit haspractical advantages for point-of-care and point-of-use diagnosticapplications.

1. A method of capturing a biological material in a fluid specimen, themethod comprising: introducing the fluid specimen containing thebiological material into a first chamber of a device, the first chambercontaining a solid phase material that has an affinity for thebiological material, the device having a first fitting attached to anentrance opening in the first chamber and a second fitting attached toan exit opening in the first chamber, the first fitting and the secondfitting having filter inserts that retain the solid phase material whileallowing rapid flow of the fluid specimen through the filter insert; andflowing the fluid specimen containing the biological material throughthe solid phase material at low pressure and rapid rate, the fluidspecimen exiting the device via the exit opening.
 2. The method of claim1 further comprising: lysing the fluid specimen, prior to introducingthe fluid specimen into the first chamber, in a second chamber fluidlyconnected to said entrance opening of said first chamber, said secondchamber comprising: a lysing medium comprising a particulate material;and an impeller, wherein said impeller is capable of impartingrotational motion to said lysing medium to mechanically lyse said fluidspecimen.
 3. The method of claim 2 wherein lysing the fluid specimencomprises agitating the specimen in the second chamber with a mediumthat includes a particulate material to mechanically lyse the specimen.4. The method of claim 1 wherein introducing a fluid specimen into thefirst chamber of a device includes introducing the specimen into a firstchamber containing a solid phase material having an affinity for atleast one of a nucleic acid, a protein, a polypeptide, a His-taggedprotein, a His-tagged polypeptide, a GST-tagged protein, a GST-taggedpolypeptide, streptavidin, biotin, Calmodulin, an antigen, an antibody,an immunoglobulin, albumin, double stranded DNA, an RNA/DNAheteroduplex, maltose binding protein, FLAG-tagged protein, FLAG-taggedpolypeptide, MYC-tagged protein, MYC-tagged polypeptide MYC, aglycoprotein, a SNAP-tag, an enzyme, a lipid-containing biologicalmaterial, a glycosylated protein, a phosphorylated protein, and amicroorganism having a high cell wall lipid content.
 5. The method ofclaim 1 wherein introducing a fluid specimen into the first chamber of adevice includes introducing the specimen into a first chamber containinga solid phase material having an affinity for more than one biologicalmaterial.
 6. The method of claim 1 wherein introducing a fluid specimeninto the first chamber of a device includes introducing the specimeninto a first chamber containing a solid phase material comprising aparticulate or a bead.
 7. The method of claim 6 wherein introducing afluid specimen into the first chamber of a device includes introducingthe specimen into a first chamber containing a solid phase materialcomprising a particulate or bead having a diameter or lateral dimensionof at least 10 μm.
 8. The method of claim 1 wherein introducing a fluidspecimen into the first chamber of a device having a fitting having afilter insert includes introducing the specimen into a device having afilter insert of pore size between about 10 μm and about 200 μm.
 9. Themethod of claim 1 wherein introducing a fluid specimen into the firstchamber of a device having a first fitting and a second fitting havingfilter inserts includes introducing the specimen into a device having awire mesh insert.
 10. The method of claim 9 wherein introducing a fluidspecimen into the first chamber of a device having a first fitting and asecond fitting having filter inserts includes introducing the specimeninto a device having a stainless steel mesh insert.
 11. The method ofclaim 1 wherein introducing a fluid specimen into the first chamber of adevice having a first fitting and a second fitting having filter insertsincludes introducing the specimen into a device having a plastic meshinsert.
 12. The method of claim 1, further comprising: eluting thebiological material captured on the solid phase material by flowing anelution medium through the solid phase at low pressure and rapid rate;and collecting an effluent containing the biological material, theeffluent exiting the device via the exit opening.
 13. A device forcapturing a biological material from a fluid specimen at rapid flow rateand low pressure, the device comprising: a first chamber containing asolid phase material that has an affinity for the biological material; afirst fitting attached to an inlet opening of the first chamber, thefirst fitting having a first thin filter insert that retains the solidphase material while allowing rapid flow of the fluid specimen at lowpressure; and a second fitting attached to an outlet opening of thefirst chamber, the second fitting having a second thin filter insertthat retains the solid phase material while allowing rapid flow of thefluid specimen at low pressure.
 14. The device of claim 13 furthercomprising: a second chamber fluidly connected to said entrance openingof said first chamber, said second chamber comprising: a lysing mediumcomprising a particulate material; and an impeller, wherein saidimpeller is capable of imparting rotational motion to said lysing mediumto mechanically lyse said fluid specimen.
 15. The device of claim 13wherein the solid phase material has an affinity for at least one of anucleic acid, a protein, a polypeptide, a His-tagged protein, aHis-tagged polypeptide, a GST-tagged protein, a GST-tagged polypeptide,streptavidin, biotin, Calmodulin, an antigen, an antibody, animmunoglobulin, albumin, double stranded DNA, an RNA/DNA heteroduplex,maltose binding protein, FLAG-tagged protein, FLAG-tagged polypeptide,MYC-tagged protein, MYC-tagged polypeptide MYC, a glycoprotein, aSNAP-tag, an enzyme, a lipid-containing biological material, aglycosylated protein, a phosphorylated protein, and a microorganismhaving a high cell wall lipid content.
 16. The device of claim 13wherein the solid phase material has an affinity for more than onebiological material.
 17. The device of claim 13 wherein the solid phasematerial is a particulate or a bead.
 18. The device of claim 17 whereinthe particulate or bead has a diameter or lateral dimension of at least10 μm.
 19. The device of claim 13 wherein the first and second filterinserts have a pore size between about 10 μm and about 200 μm.
 20. Thedevice of claim 13 wherein the filter inserts are wire mesh inserts. 21.The device of claim 20 wherein the wire mesh inserts are stainlesssteel.
 22. The device of claim 13 wherein the filter inserts are plasticmesh.